Les Cupressacées

[1] - Laurent J, Guinnepain MT, Lafay M, Sauvaget J. La pollinose des arbres. Rev Fr Allergol Immunol Clin 1999;39:276-282

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[2] - di Felice G, Barletta B, Tinghino R, Pini C. Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens. Int Arch Allergy Immunol 2001;125:280-289

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity

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[3] - Charpin D, Calleja M, Lahoz C, Pichot C, Waisel Y. Allergy to cypress pollen. Allergy 2005;60:293-301

Although Cupressus sempervirens has been spread over southern Europe since antiquity, cypress pollen allergy has not been reported until 1945. In France, the very first case reports were published in 1962. Since then, the prevalence of cypress pollinosis seems to demonstrate an upward trend, concomitantly with the increased use of cypress trees as ornamental plants, as wind breaks and as hedges. Hyposensitization, using improved pollen extracts, is increasingly prescribed. Besides, prevention measures begin to be implemented. Such measures include avoidance of planting new cypress trees, especially near human populations' centres, trimming of cypress hedges before the pollination season and agronomical research for hypoallergenic trees. Altogether, such new developments in cypress allergy deserve an update review.

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[4] - Ariano R. Allergie aux pollens de Cyprès. Rev Fr Allergol Immunol Clin 2008;48:321-324

L‚allergie au pollen des Cupressacées dans ces dernières décennies a développé sa présence dans la zone méditerranéenne. Cette allergie constitue un des rares cas de pollinose hivernale. En ce qui concerne les manifestations cliniques, elles sont des plus nombreuses rhinites et des conjonctivites par rapport à l‚asthme, il y a eu une augmentation progressive des concentrations totales et annuelles de pollens de Cupressacées. La raison de cet accroissement est due à l‚accroissement parallèle des plantations de ces arbres et il est même dû aux modifications du climat et l‚effet de serre qui augmentent la production des pollens et leur concentration dans l‚air ambiant. L‚efficacité et la sûreté de l‚immunothérapie spécifique dans la pollinose aux Cupressacées ont été démontrées depuis longtemps par des études contrôlées en double insu, soit avec la thérapie traditionnelle sous-cutanée, soit avec la thérapie sublinguale.

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[5] - Ariano R, Antico A, Di Lorenzo G, Artesani MC, Bagnato G, Bonadonna P, et al. An Epidemiological Survey of Cupressaceae Pollenosis in Italy. J Investig Allergol Clin Immunol 2003;12:287-292

Allergic reaction to Cupressaceae and Taxodiaceae pollens appears to be on the increase in the last years in Italy. An epidemiological survey on this pollenosis was conducted in 12 Italian centers. The diagnostic panel consisted of seven Cupressaceae and Taxodiaceae genus (Cupressus sempervirens, Cupressus arizonica, Cryptomeria japonica, Chamaecyparis obtusa, Thuja orientalis, Taxus baccata, and Juniperus oxycedrus). A total of 3057 pollen-sensitized outpatients were examined. The study took place from November 1999 to March 2000. At the same time pollen counts were carried out. The prevalence of positive skin tests to the diagnostic panel is different in northern (9.2%), central (28.2%), and southern (20.1%) Italy, the general average being 18.4%. The average age of sensitized patients was 36.99. Monosensitized patients represent only 14.7% of total Cupressaceae-sensitized patients, their average age being higher than the polysensitized ones (43.3 versus 35.86). Distribution of symptoms is as follows: rhinitis (49%), conjunctivitis (32%), asthma (16%), and dermatitis (3%). Months with the highest levels of symptoms are February and March. The more frequent allergens at prick tests are Cupressus sempervirens (90%) and Cupressus arizonica (88.9%). The more reactive allergens are the Cupressus arizonica and Juniperus oxycedrus. The clinical data suggest that, in Italy at least, this pollenosis is on the increase. The use of more than one extract of Cupressaceae and Taxodiaceae genus increases the diagnostic sensitivity of the disease.

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[6] - Suárez-Cervera M, Castells T, Vega-Maray A, Civantos E, del Pozo V, Fernández-González D, et al. Effects of air pollution on Cup a 3 allergen in Cupressus arizonica pollen grains. Ann Allergy Asthma Immunol 2008;101:57-66

BACKGROUND: Cupressaceae is a family of plants resistant to airborne contamination, and its pollen is the main cause of winter allergic respiratory diseases, especially in North America, Japan, and Mediterranean countries. Recently, a major allergen from Cupressus arizonica pollen grains, Cup a 3, was cloned and expressed. OBJECTIVE: To study the effects of air pollution on the expression of Cup a 3, a thaumatinlike protein, in C. arizonica pollen grains using a combination of transmission electron microscopy and immunocytochemical techniques. METHODS: Observations were made in mature and hydrated C. arizonica pollen grains from various regions in Spain with different degrees of air pollution. Specimens were fixed using freezing protocols, and ultrathin sections were incubated with anti-rCup a 3 rabbit polyclonal antibodies. RESULTS: Labeling of Cup a 3 was detected in mature and hydrated C. arizonica pollen grains. It was more intense in pollen from polluted air regions, and abundant gold particles were observed as they were released through the pollen grain walls. Furthermore, gold particles remained abundant in the pollen cytoplasm. The labeling was noticeably lower in pollen grains from unpolluted air regions. CONCLUSIONS: Cup a 3 is present in the cytoplasm and walls of cypress pollen grains during the air dispersion and hydration stages. The abundance of Cup a 3 in pollen grains under polluted air conditions indicates that these cypresses intensify their activity as a defense from environmental pollution, thus strengthening their allergenicity.

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[7] - Papa G, Romano A, Quaratino D, Di Fonso M, Viola M, Artesani MC, et al. Prevalence of sensitization to Cupressus sempervirens: a 4-year retrospective study. Sci Total Environ 2001;270(1-3):83-87

In the last few years Cupressus sempervirens has been identified as the cause of an increasing number of cases of late winter-early spring pollinosis in Mediterranean countries. In Latium, a region in central Italy, 1397 residents with complaints related to upper- or lower-respiratory-tract disorders or conjunctival disease, 243 subjects (17.4%) were skin prick positive to C. sempervirens extract, and 47 (19.3%) of this group were monosensitized. All the subjects monosensitized to cypress pollen had symptoms from January through April. Sensitization to C. sempervirens has increased from 7.2% in 1995 to 22% in 1998.

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[8] - Panzani RC. Historique de l'allergie au pollen de cyprès. Allerg Immunol (Paris) 2000;32:142-144

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[9] - Kusunoki T, Miyanomae T, Inoue Y, Itoh M, Yoshioka T, Okafuji I, et al. [Changes in Japanese cedar sensitization rates of Japanese allergic children during the last 15 years]. Arerugi 2004;53:1066-1070

In order to evaluate the changes in Japanese Cedar (JC) sensitization rates of allergic children, serum samples from 88 patients about 15 years ago (past group) and those from 91 current patients (present group) were randomly selected, and their JC specific IgE were measured with the CAP-RAST system. Sensitivity rate (class 2 or more) for JC of the present group was 65.9%, which was significantly higher than that of the past group, which was 46.6%. However, there was no significant difference between these two groups for children aged 6 or younger. For children aged 7 or older, the sensitivity rate of the present group was significantly higher than that of the past group. Thus, protection against JC sensitization, especially during early childhood, should be given serious attention.

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[10] - Charpin D, Calleja M, Lahoz C, Pichot C, Waisel Y. Allergy to cypress pollen. Allergy 2005;60:293-301

Although Cupressus sempervirens has been spread over southern Europe since antiquity, cypress pollen allergy has not been reported until 1945. In France, the very first case reports were published in 1962. Since then, the prevalence of cypress pollinosis seems to demonstrate an upward trend, concomitantly with the increased use of cypress trees as ornamental plants, as wind breaks and as hedges. Hyposensitization, using improved pollen extracts, is increasingly prescribed. Besides, prevention measures begin to be implemented. Such measures include avoidance of planting new cypress trees, especially near human populations' centres, trimming of cypress hedges before the pollination season and agronomical research for hypoallergenic trees. Altogether, such new developments in cypress allergy deserve an update review.

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[11] - Ariano R, Antico A, Di Lorenzo G, Artesani MC, Bagnato G, Bonadonna P, et al. An Epidemiological Survey of Cupressaceae Pollenosis in Italy. J Investig Allergol Clin Immunol 2003;12:287-292

Allergic reaction to Cupressaceae and Taxodiaceae pollens appears to be on the increase in the last years in Italy. An epidemiological survey on this pollenosis was conducted in 12 Italian centers. The diagnostic panel consisted of seven Cupressaceae and Taxodiaceae genus (Cupressus sempervirens, Cupressus arizonica, Cryptomeria japonica, Chamaecyparis obtusa, Thuja orientalis, Taxus baccata, and Juniperus oxycedrus). A total of 3057 pollen-sensitized outpatients were examined. The study took place from November 1999 to March 2000. At the same time pollen counts were carried out. The prevalence of positive skin tests to the diagnostic panel is different in northern (9.2%), central (28.2%), and southern (20.1%) Italy, the general average being 18.4%. The average age of sensitized patients was 36.99. Monosensitized patients represent only 14.7% of total Cupressaceae-sensitized patients, their average age being higher than the polysensitized ones (43.3 versus 35.86). Distribution of symptoms is as follows: rhinitis (49%), conjunctivitis (32%), asthma (16%), and dermatitis (3%). Months with the highest levels of symptoms are February and March. The more frequent allergens at prick tests are Cupressus sempervirens (90%) and Cupressus arizonica (88.9%). The more reactive allergens are the Cupressus arizonica and Juniperus oxycedrus. The clinical data suggest that, in Italy at least, this pollenosis is on the increase. The use of more than one extract of Cupressaceae and Taxodiaceae genus increases the diagnostic sensitivity of the disease.

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[12] - Cortegano I, Civantos E, Aceituno E, del Moral A, Lopez E, Lombardero M, et al. Cloning and expression of a major allergen from Cupressus arizonica pollen, Cup a 3, a PR-5 protein expressed under polluted environment. Allergy 2004;59:485-490

BACKGROUND: This paper describes the cloning and expression of the Cupressus arizonica pollen protein Cup a 3. In addition, we present its modulation under polluted environmental conditions. Species of the Cupressaceae family are important because of their high sensitization prevalence . METHODS: Cup a 3 cloning is based on the sequence of the homologous protein Jun a 3. Cup a 3 was expressed with good yield in the methylotropic yeast Pichia pastoris . RESULTS: Recombinant Cup a 3 (rCup a 3) contains 199 amino acids, 10 potential phosphorylation sites and no glycosylation sites. By immunoblot 63% of cypress allergic patients had specific immunoglobulin E antibodies against rCup a 3 (n = 104). This major allergen is homologous to members of the pathogenesis-related proteins (PR-5 group) and contributes to the overall allergenicity of C. arizonica pollen. Our results show that the increased expression of Cup a 3 is dependent on the pollution in the area where the pollen has been collected, being higher under polluted conditions . CONCLUSIONS: Cup a 3 is a PR-5 protein derived from C. arizonica pollen. The expression of the protein under polluted conditions has a direct incidence on the pollen allergenicity, as has been demonstrated by skin tests and Radioallergosorbent test inhibition.

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[13] - Lopes Silva S, Rodrigues Alves R, Spínola Santos A, Pregal A, Mendes A, Pedro E, et al. Cypress Allergy in Portugal. J Allergy Clin Immunol 2005;115(2 suppl.):S238

RATIONALE: Since cypress allergy has been claimed as a major cause of winter rhinoconjuntivitis and asthma in the Mediterranean area, we wanted to evaluate if this extract should be included in our Unit‚s standard panel of allergens METHODS: We studied 105 sequential patients with respiratory allergy Prick tests to Cupressus sempervirens (Cs), Cupressus arizonica (Ca) and Juniperus ashei (Ja) were performed along with a standard panel of allergens including grass mix, parietaria, ragweed, plantain, olive, platanus, dust mite, cat and dog dander and fungi. Specific IgE (sIgE) was performed only in selected cases RESULTS: Among the 105 patients evaluated (65F/40M; mean age: 32.0±14.8 years), 45.7% had rhinitis, 1.9% asthma and 52.3% had both diseases. Cutaneous sensitization to cupressaceae (21,9%), was the fourth most prevalent pollen sensitization after grasses (49.5%), olive (24.8%) and parietaria (22.8%). Comparing patients sensitized to cupressaceae (C+) with non-sensitized (C-), no significant differences were found concerning sex or age distribution, clinical manifestations, total IgE or family history of atopy. Co-sensitization prevalences comparison only presented a significant statistic difference concerning Platanus (21.7% of C+ and 2.4% of C-, p<0.05). sIgE to Cs was performed in 16 patients with cutaneous sensitization and was positive in 5. Only 2 patients with cutaneous sensitization had winter related symptoms, both with indetectable sIgE CONCLUSION: Cypress cutaneous sensitization is frequent and the extract should be added to our standard panel of allergens. Specific IgE to Ca and Ja currently being performed in our population may help to establish cypress allergy real clinical relevance, still not clearly demonstrated

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[14] - Varela S, Subiza J, Subiza JL, Rodriguez R, Garcia B, Jerez M, et al. Platanus pollen as an important cause of pollinosis. J Allergy Clin Immunol 1997;100:748-754

OBJECTIVE: The existence of Platanus pollinosis is not generally accepted despite the production of very large quantities of airborne Platanus pollen in many cities of the United States and Europe. The aim of this study was to investigate if Platanus pollen really contributes to the symptoms of the patients with pollinosis in the Madrid area. METHODS: We carried out systematic skin prick testing with Platanus pollen extract on 47 patients seen in our allergy center with spring-summer pollinosis symptoms. Each patient maintained symptom score diaries before, during, and after the Platanus pollination season. The average symptom scores were calculated and compared with the Platanus pollen counts. Measurements of specific IgE by ELISA and immunoblotting also were performed in each patient. RESULTS: The Platanus skin tests were positive in 33 of the 39 patients first seen with seasonal symptoms during Platanus pollen season and only in three of the eight patients without symptoms during Platanus exposure (Fisher's exact test; p < 0.05). Twenty-two of the 33 Platanus-positive skin test patients also had a positive ELISA result. Furthermore, the average 24-hour rhinitis symptom scores of the 39 patients first seen with seasonal symptoms during March through April showed significant correlation with Platanus pollen counts (r(s) = 0.57, p < 0.05). The immunoblot results suggest that a 17 kd pollen protein could be a major allergen in patients with Platanus pollinosis. CONCLUSIONS: Platanus pollen is an important cause of pollinosis in Madrid. A protein with a molecular weight of 17 kd appeared to be its major allergen.

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[15] - Bousquet PJ, Gallega MP, Dhivert-Donnadieu H, Demoly P. Latex is not essential in a standardized skin prick test battery. Allergy 2005;60:407-408

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[16] - Hemery ML, Verdier R, Daban P, Sellier N, Dujols P, Demoly P. Sensibilisation aux gants en latex poudrés: une prévalence élevée chez le personnel hospitalier. Presse Med 2005;34:1363-1369

INTRODUCTION: The prevalence of latex allergies in industrial countries has skyrocketed since the 1980s. Between 2.6 and 22% of hospital workers are diagnosed with latex allergy, which has been recognized as an occupational hazard in France since 1997. AIM: To assess the prevalence of latex allergy among Montpellier University Hospital Center staff . METHODS: From 1999 to 2002, we interviewed and conducted skin-prick tests on 537 hospital staff members from different departments and with different degrees of exposure to airborne latex allergens . RESULTS: Symptoms while using natural-rubber latex gloves (most often contact dermatitis) were reported by 88 (16.4%) staff members. Overall work-related allergic symptoms included rhinitis, reported by 65 (12.1%), contact urticaria by 28 (5.2%), and bronchial symptoms by 16 (1.1%). In all, sensitization to latex was identified in 7.1% of our staff, and this percentage was higher (11.3%) in units where latex gloves were used more often. Sensitization was associated with high latex exposure and atopy . CONCLUSION: This high rate of latex sensitization and the risk factors are similar to those already published. Based on this study, we have eliminated powdered latex gloves, as have many other hospitals.

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[17] - Hrabina M, Dumur JP, Sicard H, Viatte A, André C. Diagnosis of cypress allergy: in vivo and in vitro standardization of a Juniperus ashei pollen extract. Allergy 2003;58:808-813

BACKGROUND: Cypress pollen allergy is a major cause of rhinoconjunctivitis and asthma in the Mediterranean area. The nonstandardized cypress allergen extracts currently available for the diagnosis of cypress allergy have a low level of activity. The search for an active material has led to the selection of Juniperus ashei (Ja) pollen because of its very high cross-reactivity with cypress extracts and its superior allergenic activity. The aim of this study was to characterize in vitro and calibrate in vivo an in-house reference extract (IHRS) of J. ashei pollen and determine the specificity and sensitivity of a standardized Ja extract for the prick test diagnosis of cypress allergy . METHODS: Juniperus ashei pollen extract was analysed by 2-D electrophoresis. The IHRS Ja extract was calibrated by skin prick testing in 28 cypress-allergic patients. The sensitivity and specificity of cypress allergy diagnosis using a standardized Ja extract was studied by skin prick test in 42 cypress-allergic patients and 53 nonallergic patients. Jun a 1 content of the IHRS was determined by a monoclonal antibody-based electrophoretic technique . RESULTS: The Jun a 1 content of the 100 IR/ml Ja IHRS extract was 180 microg/ml. For in vivo diagnosis of cypress allergy, Ja pollen extract demonstrated a sensitivity of 95%, a specificity of 100%, a negative predictive value of 96%, and a positive predictive value of 100% . CONCLUSION: Standardized Ja pollen extract is therefore a very appropriate tool for the in vivo diagnosis of cypress pollen allergy and good candidate for specific immunotherapy.

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[18] - Togawa A, Panzani RC, Garza MA, Kishikawa R, Goldblum RM, Midoro-Horiuti T. Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity. Ann Allergy Asthma Immunol 2006;97:336-342

BACKGROUND: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. OBJECTIVE: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. METHODS: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. RESULTS: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. CONCLUSION: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.

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[19] - Laaidi K, Besancenot JP, Carli PM. Evolution de l'éosinophilie au cours de la saison pollinique dans une population générale: un outil pour déterminer de nouvelles sensibilisations. Allerg Immunol (Paris) 2002;34:13-18

The purpose of this paper was to verify the effect of pollen peaks on blood eosinophilia in an all and sundry population, including allergic as well as non-allergic subjects, so that we can open up new horizons in the understanding and prevention of pollinosis. Daily eosinophilia counts of hospital patients were measured at the time of a blood checkup (1996-1998), and divided into six classes. Those data were compared to daily pollen counts of twelve taxa, coming from the Hirst trap of Dijon (France). An eosinophilia increase occurred when hazel, hornebeam, birch, oak, grasses, ragweed and plantain were present in high concentration. In other cases, only simultaneous presence of several taxa seemed to play a part, because of cross-reactivity or polysensitization. Lastly, Cupressaceae-Taxaceae and ragweed were seen as increasing eosinophilia in seemingly non allergic people. The analysis of eosinophilia in the general population was able to reveal potential allergic patients and potential allergic diseases.

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[20] - Agea E, Bistoni O, Russano A, Corazzi L, Minelli L, Bassotti G, et al. The biology of cypress allergy. Allergy 2002;57:959-960

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[21] - Papa G, Romano A, Quaratino D, Di Fonso M, Viola M, Artesani MC, et al. Prevalence of sensitization to Cupressus sempervirens: a 4-year retrospective study. Sci Total Environ 2001;270(1-3):83-87

In the last few years Cupressus sempervirens has been identified as the cause of an increasing number of cases of late winter-early spring pollinosis in Mediterranean countries. In Latium, a region in central Italy, 1397 residents with complaints related to upper- or lower-respiratory-tract disorders or conjunctival disease, 243 subjects (17.4%) were skin prick positive to C. sempervirens extract, and 47 (19.3%) of this group were monosensitized. All the subjects monosensitized to cypress pollen had symptoms from January through April. Sensitization to C. sempervirens has increased from 7.2% in 1995 to 22% in 1998.

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[22] - Ariano R, Antico A, Di Lorenzo G, Artesani MC, Bagnato G, Bonadonna P, et al. An Epidemiological Survey of Cupressaceae Pollenosis in Italy. J Investig Allergol Clin Immunol 2003;12:287-292

Allergic reaction to Cupressaceae and Taxodiaceae pollens appears to be on the increase in the last years in Italy. An epidemiological survey on this pollenosis was conducted in 12 Italian centers. The diagnostic panel consisted of seven Cupressaceae and Taxodiaceae genus (Cupressus sempervirens, Cupressus arizonica, Cryptomeria japonica, Chamaecyparis obtusa, Thuja orientalis, Taxus baccata, and Juniperus oxycedrus). A total of 3057 pollen-sensitized outpatients were examined. The study took place from November 1999 to March 2000. At the same time pollen counts were carried out. The prevalence of positive skin tests to the diagnostic panel is different in northern (9.2%), central (28.2%), and southern (20.1%) Italy, the general average being 18.4%. The average age of sensitized patients was 36.99. Monosensitized patients represent only 14.7% of total Cupressaceae-sensitized patients, their average age being higher than the polysensitized ones (43.3 versus 35.86). Distribution of symptoms is as follows: rhinitis (49%), conjunctivitis (32%), asthma (16%), and dermatitis (3%). Months with the highest levels of symptoms are February and March. The more frequent allergens at prick tests are Cupressus sempervirens (90%) and Cupressus arizonica (88.9%). The more reactive allergens are the Cupressus arizonica and Juniperus oxycedrus. The clinical data suggest that, in Italy at least, this pollenosis is on the increase. The use of more than one extract of Cupressaceae and Taxodiaceae genus increases the diagnostic sensitivity of the disease.

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[23] - Bistoni O, Emiliani C, Agea E, Russano AM, Mencarelli S, Orlacchio A, et al. Biochemical and Immunological Characterization of Pollen-Derived ß-Galactosidase Reveals a New Cross-Reactive Class of Allergens among Mediterranean Trees. Int Arch Allergy Immunol 2005;136:123-133

BACKGROUND: The most potent allergens in the Spermatophytae family exhibit significant homology with enzymes. Some of these are though to be involved in pectin metabolism, recognition of compatible stigma and delivery of sperm cells to the ovule . OBJECTIVE: To test if glycohydrolase activities from some Mediterranean tree pollens could act as allergens in sensitized hosts . METHODS: Freshly collected Cupressus and Olea pollens were investigated for their glycohydrolase activities by means of synthetic fluorogenic substrates and isoenzymes characterized by DEAE-cellulose ion-exchange chromatography. Binding of specific IgE was investigated by immunoblotting in 30 tree-sensitive subjects, as well as in 20 atopic non-tree-sensitive and 15 healthy controls. The enzymes were also adopted to stimulate proliferation of allergen-specific T cell clones. Finally, they were tested in vivo in a cutaneous immediate wheal and flare reaction . RESULTS: beta-Galactosidase (beta-GAL) is present with different isoenzymatic patterns on both pollen extracts, could be recognized by circulating IgE, as well as immunoprecipitated by sera from allergic subjects. The enzyme could stimulate the proliferation of T cells from allergic subjects, and favor the emergence of CD4+ T cell clones with specific in vitro reactivity to beta-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects . CONCLUSIONS: Despite different isoenzymatic patterns, Olea-derived beta-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.

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[24] - Charpin D. Epidemiology of cypress allergy. Allerg Immunol (Paris) 2000;32:83-85

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[25] - Bousquet J, Cour P, Guerin B, Michel FB. Allergy in the Mediterranean area I. Pollen counts and pollinosis of Montpellier. Clin Allergy 1984;14:249-258

The climatic conditions of the Mediterranean area result in vegetation and pollen very different from that of the other parts of Europe. The pollen content of the atmosphere of Montpellier, southern France, was examined using a filter sampler which was shown to be more efficient than most of the current devices for air sampling. Pollen counts were subsequently compared with pollinosis of patients born and living in and around Montpellier. The mean annual pollen counts showed that grass pollens and Cupressaceae pollens (cypress and juniper) are the highest. Some Mediterranean pollens (Oleaceae, London plane, Parietaria) are also important. Plantain and oak pollens are also present in relatively large amounts. Grass pollen allergy was found to be present in 86.5% of pollen-allergic patients. It was followed by plantain, Parietaria, Oleaceae, London plane and Cupressaceae pollens which were allergenic in 13-36% of pollen-allergic patients. Oak and pine pollens were present in large quantities in the counts but few persons were sensitive to oak and none to pine. By contrast, some patients had positive skin tests to alfalfa, red clover, acacia and lime tree pollens though these pollens were almost absent from the counts. In a few cases local sources of these pollens could account for the positive skin tests but cross-sensitivities could also occur. In summary, pollinosis of the Northern Mediterranean area is intermediate between the southern part of the area and the other parts of Europe.

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[26] - Boutin-Forzano S, Gouitaa M, HammouY, Ramadour M, Charpin D. Personal risk factors for cypress pollen allergy. Allergy 2005;60:533-535

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[27] - Gunawan H, Takai T, Ikeda S, Okumura K, Ogawa H. Protease Activity of Allergenic Pollen of Cedar, Cypress, Juniper, Birch, and Ragweed. Allergol Int 2008;57:83-91

Background: Pollen is an important trigger of allergic rhinitis, conjunctivitis, and/or asthma, and an exacerbating factor in atopic dermatitis. Although it is proposed that protease activity from allergen sources, such as mites, enhances allergenicity, little information is available on that from relevant allergenic pollens such as Japanese cedar and Japanese cypress pollens, which are the major cause of pollinosis in Japan. Methods: We analyzed the protease activities derived from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and white birch and short ragweed, using synthetic substrates and class-specific inhibitors. Results: We found that the pollen of the three members of the Cupressaceae/Taxodiaceae family contained serine protease activity, that the pollen of white birch and short ragweed contained not only serine protease activity but also cysteine protease activity, that all five types of pollen tested contained at least one other type of serine protease, whose sensitivity to a serine protease-specific inhibitor was relatively low, and that the content and releasability of the pollen-derived proteases differed according to the plant families. Conclusions: Clinically relevant allergenic pollens tested in the present study can release serine and/or cysteine endopeptidases. Information on the spectrum of the endopeptidase activities from these allergenic pollen grains will be useful for investigating their contribution to the pathogenesis of allergies.

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[28] - Mothes N, Westritschnig K, Valenta R. Tree pollen allergens. Clin Allergy Immunol 2004;18:165-184

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[29] - Yasueda H, Yui Y, Shimizu T, Shida T. Isolation and partial characterization of the major allergen from Japanese cedar (Cryptomeria japonica) pollen. J Allergy Clin Immunol 1983;71:77-86

A purified allergen, antigen SBP, was obtained from the pollen of the Japanese cedar (Cryptomeria japonica, Sugi in Japanese) by ammonium sulfate precipitation, ion-exchange chromatography on diethylaminoethyl and carboxymethyl cellulose, and gel chromatography on Sephadex G-150. Antigen SBP was a heat-sensitive basic glycoprotein of approximately 40,000 molecular weight. By preparative isoelectric focusing and gel chromatography on Sephadex G-100, antigen SBP could be further separated into four subfractions, differing in both isoelectric point and molecular weight. By immunodiffusion analysis, direct skin testing, and radioallergosorbent test inhibition, it was shown that antigen SBP was the major allergen of Japanese cedar pollen, and the four subfractions were seen to be antigenically and allergenically identical.

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[30] - Fujimura T, Shigeta S, Kawamoto S, Aki T, Masubuchi M, Hayashi T, et al. Two-Dimensional IgE-Binding Spectrum of Japanese Cedar (Cryptomeria japonica) Pollen Allergens. Int Arch Allergy Immunol 2004;133:125-135

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen . OBJECTIVES: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis . METHODS: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens . RESULTS: 2-D immunolabeling with individual patients' sera showed the distinguishable IgE-binding patterns inlaid with 4-87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5-75% of IgE-binding frequency) and 3 Cry j 2 (32.5-40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2 . CONCLUSIONS: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.

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[31] - Fujimura T, Shigeta S, Kawamoto S, Aki T, Masubuchi M, Hayashi T, et al. Two-Dimensional IgE-Binding Spectrum of Japanese Cedar (Cryptomeria japonica) Pollen Allergens. Int Arch Allergy Immunol 2004;133:125-135

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen . OBJECTIVES: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis . METHODS: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens . RESULTS: 2-D immunolabeling with individual patients' sera showed the distinguishable IgE-binding patterns inlaid with 4-87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5-75% of IgE-binding frequency) and 3 Cry j 2 (32.5-40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2 . CONCLUSIONS: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.

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[32] - Fujimura T, Shigeta S, Kawamoto S, Aki T, Masubuchi M, Hayashi T, et al. Two-Dimensional IgE-Binding Spectrum of Japanese Cedar (Cryptomeria japonica) Pollen Allergens. Int Arch Allergy Immunol 2004;133:125-135

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen . OBJECTIVES: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis . METHODS: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens . RESULTS: 2-D immunolabeling with individual patients' sera showed the distinguishable IgE-binding patterns inlaid with 4-87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5-75% of IgE-binding frequency) and 3 Cry j 2 (32.5-40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2 . CONCLUSIONS: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.

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[33] - Czerwinski EW, Midoro-Horiuti T, White MA, Brooks EG, Goldblum RM. Crystal Structure of Jun a 1, the Major Cedar Pollen Allergen from Juniperus ashei Reveals a Parallel beta-Helical Core. J Biol Chem 2005;280:3740-3746

Pollen from cedar and cypress trees is a major cause of seasonal hypersensitivity in humans in several regions of the Northern Hemisphere. We report the first crystal structure of a cedar allergen, Jun a 1, from the pollen of the mountain cedar Juniperus ashei (Cupressaceae). The core of the structure consists primarily of a parallel beta-helix, which is nearly identical to that found in the pectin/pectate lyases from several plant pathogenic microorganisms. Four IgE epitopes mapped to the surface of the protein are accessible to the solvent. The conserved vWiDH sequence is covered by the first 30 residues of the N terminus. The potential reactive arginine, analogous to the pectin/pectate lyase reaction site, is accessible to the solvent, but the substrate binding groove is blocked by a histidine-aspartate salt bridge, a glutamine, and an alpha-helix, all of which are unique to Jun a 1. These observations suggest that steric hindrance in Jun a 1 precludes enzyme activity. The overall results suggest that it is the structure of Jun a 1 that makes it a potent allergen.

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[34] - Aceituno E, Del Pozo V, Minguez A, Arrieta I, Cortegano I, Cardaba B, et al. Molecular cloning of major allergen from Cupressus arizonica: Cup a1. Clin Exp Allergy 2000;30:1750-1758

The family Cupressaceae is a relevant source of allergens that causes winter respiratory allergies. Cloning and sequencing the major antigen of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients. To obtain a full-length complementary DNA for Cup a 1, the major allergen of Cupressus arizonica pollen. It was cloned and sequenced and the recombinant protein was expressed. Messenger RNA from Cupressus arizonica pollen was obtained and the Cup a 1 sequence was established using a 3'-RACE system and primers based on the N-terminal amino acid sequence. Recombinant Cup a 1 was cloned in pBluescript and expressed in a glycosylated form in rabbit reticulocytes. The cDNA was subcloned in pGEX-5X-1 and expressed in Escherichia coli as a fusion protein with GST. Recombinant Cup a 1 is highly homologous with the major allergens of mountain cedar (Jun a 1), Japanese cypress (Cha o 1) and Japanese cedar (Cry j 1). Cup a 1 contains three potential N-glycosylation sites that are different from those found in Jun a 1 and Cry j 1. The cloned protein contains a pectate lyase active site identical to those of Cry j 1 and Jun a 1. The IgE from patients' sera recognizes recombinant Cup a 1, and this reactivity is higher with the glycosylated protein. Cup a 1 has been cloned and sequenced. As expected, the high degree of homology with Cha o 1, Jun a 1 and Cry j 1 explains the cross-reactivity of conifer pollens. Different IgE reactivity with the glycosylated and non-glycosylated protein suggests the importance of carbohydrate moieties in the IgE binding site.

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[35] - Arilla MC, Ibarrola I, Garcia R, de la Hoz B, Martinez A, Asturias JA. Quantification of the Major Allergen from Cypress (Cupressus arizonica) Pollen, Cup a 1, by Monoclonal Antibody-Based ELISA. Int Arch Allergy Immunol 2004;134:10-16

BACKGROUND: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract . METHODS: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques . RESULTS: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5-1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity . CONCLUSIONS: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.

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[36] - Midoro-Horiuti T, Goldblum RM, Kurosky A, Wood TG, Schein CH, Brooks EG. Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:613-617

BACKGROUND: Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1 . OBJECTIVE: A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed . METHODS: Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera . RESULTS: Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis . CONCLUSION: The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.

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[37] - Midoro-Horiuti T, Schein CH, Mathura V, Braun W, Czerwinski EW, Togawa A, et al. Structural basis for epitope sharing between group 1 allergens of cedar pollen. Mol Immunol 2006;43:509-518

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

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[38] - di Felice G, Barletta B, Tinghino R, Pini C. Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens. Int Arch Allergy Immunol 2001;125:280-289

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity

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[39] - Sone T, Komiyama N, Shimizu K, Kusakabe T, Morikubo K, Kino K. Cloning and sequencing of cDNA coding for Cry j I, a major allergen of Japanese cedar pollen. Biochem Biophys Res Commun 1994;199:619-625

cDNA clones coding for Cry j I, a major allergen of the Japanese cedar (Cryptomeria japonica) pollen, have been isolated. Two of the clones were sequenced and found to code for a putative 21-residue signal peptide and a 353-residue mature protein with a derived molecular weight of 38.5 KDa. Five possible N-linked glycosylation sites were found in the sequence. Comparison of the nucleotide sequences of the two clones revealed sixteen nucleotide differences and these led to five amino acid exchanges in the mature allergen, indicating that an isoform of the Cry j I molecule exists. The deduced amino acid sequence of the Cry j I shows 46-48% identities with those of the Amb a I family and Amb a II, the major allergens of short ragweed. These findings should facilitate study of the structure-function relationship between the allergen and the immunocompetent cells

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[40] - Alisi C, Afferni C, Iacovacci P, Barletta B, Tinghino R, Butteroni C, et al. Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen. Allergy 2001;56:978-984

Background: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. Methods: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. Results: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF3 (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF3/GnGXF3 (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF3/Gn(GF)XF3 (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.

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[41] - Asturias JA, Diéguez M, Ibarrola I, García R, De la Hoz B, Martínez A. Purified natural and recombinant Cup a 1 for diagnosis of Cupressus arizonica sensitization. Allergy Clin Immunol Int 2005;17(Suppl. 1):204

Background: C. arizonica is used in many Mediterranean countries for ornamental purposes in gardens and parks and also as wind and noise barriers. The low protein and high carbohydrate content found in Cupressaceae pollen extracts have hindered the availability of standardized extracts for diagnosis and immunotherapy. Allergenic extract of C. arizonica pollen contains a glycoprotein of 43 kDa (Cup a 1) which have an incidence of 82% among Arizona cypress-allergic patients. OBJECTIVE: To compare C. arizonica pollen extract and purified natural and recombinant Cup a 1 for their diagnostic value and to determine the importance of carbohydrate epitopes on diagnosis. METHODS: Cup a 1 was purified from C. arizonica pollen extract by hydrophobic interaction followed by gel permeation chromatography. Cup 1 encoding cDNA was cloned by RT-PCR, sequenced and subcloned in pTrc-HisB vector. rCup a 1 was produced in Escherichia coli and purified by metal-chelating chromatography. 27 patients with C. arizonica pollen allergy and 20 control subjects, 10 no-atopics and 10 with other no-related allergies, were included in the study. Skin prick test (SPT) was performed with C. arizonica pollen extract and natural and recombinant Cup a 1 at different concentrations (0.2, 2, 20, and 200 µg/ml). RESULTS: Both natural and recombinant Cup a 1 were able to elicit positive responses in vivo and in vitro. Skin response with purified allergens (both natural and recombinant) was dependent of allergen concentration. In SPT, sensitivity and specificity of nCup a 1 at 20 µg/ml were 81.5 and 94.4%, respectively and 74.1 and 94.4% for rCup a 1. At 200 µg/ml for both Cup a 1, the sensitivity was near 100% and the specificity was 83.3%. rCup a 1 exhibited in SPT a good correlation with nCup a 1 (r = 0.53; p<0.001). The sera of 24 and 23 out of 27 allergic patients contained IgE antibodies raised against nCup a 1 or total pollen extract respectively, while rCup a 1 reacted with only 11 patient sera. These different IgE binding values between nCup a 1 and rCup a 1 persisted after deglycosylation of natural allergen by treatment with peryodate. CONCLUSION: Either purified natural and recombinant Cup a 1 are sufficient for a reliable in vivo diagnosis of C. arizonica pollen allergy in most patients and induce comparable skin prick reactivity as the whole extract.

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[42] - Fujimura T, Shigeta S, Suwa T, Kawamoto S, Aki T, Masubuchi M, et al. 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. Clin Exp Allergy 2005;35:234-243

Summary 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.

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[43] - Midoro-Horiuti T, Goldblum RM, Kurosky A, Goetz DW, Brooks EG. Isolation and characterization of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:608-612

Cedar pollens are important causes of seasonal allergic disease in diverse geographic areas. OBJECTIVE: A major allergen from mountain cedar (Juniperus ashei) pollen, termed Jun a 1, was isolated and characterized. METHODS: Water-soluble pollen glycoproteins were extracted, salt precipitated, and purified with use of concanavalin A affinity chromatography or HPLC. The purified fractions were characterized by SDS-PAGE, immunoblotting, and N-terminal amino acid sequence analysis. Binding of allergen-specific IgE from the sera of cedar-hypersensitive patients was detected by ELISA and antigen-specific responses of peripheral blood T cells by tritiated thymidine incorporation. RESULTS: The major extractable cedar pollen glycoprotein had a molecular weight and N-terminal amino acid sequence that was similar to that of the major allergen Cha o 1, from Japanese cypress (Chamaecyparis obtusa), and Cry j 1, from Japanese cedar (Cryptomeria japonica). IgE from cedar-hypersensitive patients' sera bound to the isolated glycoprotein. CONCLUSION: The predominance of Jun a 1 in the soluble proteins of mountain cedar pollen and its high degree of homology with Cha o 1 and Cry j 1 make it likely to be the major allergen of this pollen. Amino acid sequence conservation also makes Jun a 1 a potential target for cross-reactivity between these pollen allergens. The observed reactivity of IgE from the sera of Japanese cedar-sensitive patients with Jun a 1 is consistent with this proposition.

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[44] - Midoro-Horiuti T, Goldblum RM, Kurosky A, Goetz DW, Brooks EG. Isolation and characterization of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:608-612

Cedar pollens are important causes of seasonal allergic disease in diverse geographic areas. OBJECTIVE: A major allergen from mountain cedar (Juniperus ashei) pollen, termed Jun a 1, was isolated and characterized. METHODS: Water-soluble pollen glycoproteins were extracted, salt precipitated, and purified with use of concanavalin A affinity chromatography or HPLC. The purified fractions were characterized by SDS-PAGE, immunoblotting, and N-terminal amino acid sequence analysis. Binding of allergen-specific IgE from the sera of cedar-hypersensitive patients was detected by ELISA and antigen-specific responses of peripheral blood T cells by tritiated thymidine incorporation. RESULTS: The major extractable cedar pollen glycoprotein had a molecular weight and N-terminal amino acid sequence that was similar to that of the major allergen Cha o 1, from Japanese cypress (Chamaecyparis obtusa), and Cry j 1, from Japanese cedar (Cryptomeria japonica). IgE from cedar-hypersensitive patients' sera bound to the isolated glycoprotein. CONCLUSION: The predominance of Jun a 1 in the soluble proteins of mountain cedar pollen and its high degree of homology with Cha o 1 and Cry j 1 make it likely to be the major allergen of this pollen. Amino acid sequence conservation also makes Jun a 1 a potential target for cross-reactivity between these pollen allergens. The observed reactivity of IgE from the sera of Japanese cedar-sensitive patients with Jun a 1 is consistent with this proposition.

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[45] - Midoro-Horiuti T, Schein CH, Mathura V, Braun W, Czerwinski EW, Togawa A, et al. Structural basis for epitope sharing between group 1 allergens of cedar pollen. Mol Immunol 2006;43:509-518

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

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[46] - Varshney S, Goldblum RM, Auton M, Kearney C, Watanabe M, Midoro-Horiuti T. Major mountain cedar allergen, Jun a 1, contains conformational as well as linear IgE epitopes. Mol Immunol 2007;44:2781-2785

We have previously identified four linear IgE epitopes on Jun a 1, the dominant allergen in mountain cedar pollen and mapped these to the surfaces of a molecular model and to the crystal structure of this glycoprotein. The aim of the present study was to determine if Jun a 1 also displays conformational IgE epitopes. Jun a 1 was denatured by heating at 75 degrees C for 1h, exposure to 6M guanidine or by reductive alkylation in the presence and absence of guanidine. The effects of these manipulations on the binding to IgE from patients with mountain cedar hypersensitivity was evaluated by an ImmunoCAP inhibition assay, using Jun a 1-specific caps. Treatment-associated changes in the 3D-structure were assessed by dynamic light scattering and CD spectroscopy. IgE binding to native Jun a 1 was inhibited 92+/-9% by soluble native protein and 92+/-9% by reduced and alkylated Jun a 1. However, the capacity of Jun a 1 to inhibit the binding of IgE antibodies was significantly diminished upon denaturation by heat, guanidine alone, or reduction and alkylation in guanidine, compared to native Jun a 1. Reductive alkylation treatment under denaturing conditions also increased the Stoke's radius, suggesting that the protein was partially unfolded. Analysis of the circular dichroism (CD) spectra suggested that heating and treatment with guanidine caused a loss of alpha-helical structure. Guanidine also caused an increase in random coil structure. Thus, at least a portion of the anti-Jun a 1 IgE antibodies produced by allergic humans recognize conformational epitopes and it is likely that some of these epitopes reside in alpha-helical structures of Jun a 1.

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[47] - Midoro-Horiuti T, Mathura V, Schein CH, Braun W, Yu S, Watanabe M, et al. Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site. Mol Immunol 2003;40:555-562

Resolution of the 3D structures and IgE epitopes of allergens may identify common or conserved features of allergens. Jun a 1, the predominant allergen in mountain cedar pollen, was chosen as a model for identifying common structural and functional features among a group of plant allergens. In this study, synthetic, overlapping peptides of Jun a 1 and sera from patients allergic to mountain cedar pollen were used to identify linear epitopes. A 3D model of Jun a 1 was produced using the Bacillus subtiles pectate lyase (PL) as a template and validated with biophysical measurements. This allowed mappings of four IgE binding sites on Jun a 1. Two of the epitopes mapped to turns or loops on the surface of the model structure. The other two epitopes mapped to the beta-sheet region, homologous to the catalytic site of PL. This region of Jun a 1 is highly conserved in the group 1 allergens from other cedar trees as well as microbial PLs. The finding that two out of three major IgE epitopes map to highly conserved catalytic regions of group 1 cedar allergens may help to explain the high degree of cross-reactivity between cedar pollen allergens and might represent a pattern of reactivity common to other allergens with catalytic activity.

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[48] - Takagi K, Teshima R, Sawada J. Determination of Human Linear IgE Epitopes of Japanese Cedar Allergen Cry j 1. Biol Pharm Bull 2005;28:1496-1499

Cry j 1 is one of the major allergens in Japanese cedar pollen. We attempt high throughput analysis and comprehensive identification of the linear IgE epitopes of Cry j 1. A series of overlapping synthetic Cry j 1 peptides chemically spotted on cellulose membrane was probed with sera from patients in Japan and United States, which were reactive to Cry j 1, and the reactivity of one of the detected sequences was confirmed by means of competitive ELISA using peptide as coated antigen. The peptide (331)NGNATPQLTKNA(342) (peptide 166) was detected by all three pooled sera used, and peptide (103)NGGPCVFIKRVS(114) (peptide 52) was detected by two of the three pools of sera. In addition, several peptides reacted with one of the pooled sera. IgE binding to peptide 166-coated wells was inhibited by addition of peptide 166 for several individual patient sera, suggesting that peptide 166 is one of the linear epitopes of Cry j 1. Since patients in United States were suggested to be rarely sensitized with Japanese cedar, they were sensitized with the similar tree pollen allergens such as Cup s 1 and Jun a 1, and cross-reacted with Cry j 1. We have comprehensively investigated human IgE epitopes of Cry j 1 and succeeded in identifying a common linear epitope, (331)NGNATPQLTKNA(342).

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[49] - Midoro-Horiuti T, Schein CH, Mathura V, Braun W, Czerwinski EW, Togawa A, et al. Structural basis for epitope sharing between group 1 allergens of cedar pollen. Mol Immunol 2006;43:509-518

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

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[50] - Midoro-Horiuti T, Goldblum RM, Kurosky A, Wood TG, Schein CH, Brooks EG. Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1. J Allergy Clin Immunol 1999;104:613-617

BACKGROUND: Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1 . OBJECTIVE: A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed . METHODS: Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera . RESULTS: Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis . CONCLUSION: The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.

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[51] - Yokoyama M, Miyahara M, Shimizu K, Kino K, Tsunoo H. Purification, identification, and cDNA cloning of Jun a2, the second major allergen of mountain cedar pollen. Biochem Biophys Res Commun 2000;275:195-202

The second major allergen of Juniperus ashei (mountain cedar) pollen, Jun a 2, has been purified and its cDNA cloned. The purified protein has a molecular mass of 43 kDa and its N-terminal 9-residue amino acid sequence is highly homologous to those of Cry j 2 and Cha o 2, the second major allergen of Cryptomeria japonica and Chamaecyparis obtusa pollen, respectively. cDNA clones encoding Jun a 2 were isolated after PCR based amplification, and their nucleotide sequences were determined. The cDNA contains an open reading frame of 507 amino acid residues, and encodes a putative 54-residue signal sequence and a 453-residue intermediate, which releases a C-terminal fragment upon maturation. Three possible N-linked glycosylation sites and 20 cystein-residues are found in the deduced amino acid sequence. The amino acid sequence of Jun a 2 shows 70.7 and 82.0% identity with those of Cry j 2 and Cha o 2, respectively. Immunological observations that IgE antibodies in sera of Japanese pollinosis patients bind not only to Cry j 2 and Cha o 2 but also to Jun a 2 strongly suggest that Jun a 2 is an allergen of mountain cedar pollen, and that allergenic epitopes of these three allergens are similar

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[52] - Fujimura T, Shigeta S, Kawamoto S, Aki T, Masubuchi M, Hayashi T, et al. Two-Dimensional IgE-Binding Spectrum of Japanese Cedar (Cryptomeria japonica) Pollen Allergens. Int Arch Allergy Immunol 2004;133:125-135

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen . OBJECTIVES: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis . METHODS: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens . RESULTS: 2-D immunolabeling with individual patients' sera showed the distinguishable IgE-binding patterns inlaid with 4-87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5-75% of IgE-binding frequency) and 3 Cry j 2 (32.5-40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2 . CONCLUSIONS: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.

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[53] - Mori T, Yokoyama M, Komiyama N, Okano M, Kino K. Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen. Biochem Biophys Res Commun 1999;263:166-171

The second major allergen of Chamaecyparis obtusa (Japanese cypress) pollen, Cha o 2, has been purified and its cDNA cloned. Of patients with pollinosis caused by C. obtusa, 82.5% produce IgE antibodies which react with purified Cha o 2. The purified protein has a molecular mass of 46 kDa and its 12 N-terminal amino acid sequence displays a high homology with that of Cry j 2, the second major allergen of Cryptomeria japonica pollen. cDNA clones coding for Cha o 2 have been isolated using Cry j 2 cDNA as a probe. Cha o 2 cDNA clones were sequenced and found to code a putative 50-residue signal sequence and a 464-residue mature protein with a molecular weight of 50 kDa. Two possible N-linked glycosylation sites were found in the sequence. The deduced amino acid sequence of Cha o 2 shows 74.3% identity with that of Cry j 2. In its primary structure, Cha o 2 shows significant identity with those of the polygalacturonases of avocado, tomato, and maize as well as Cry j 2.

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[54] - Fujimura T, Shigeta S, Suwa T, Kawamoto S, Aki T, Masubuchi M, et al. 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. Clin Exp Allergy 2005;35:234-243

Summary 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.

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[55] - Sakaguchi M, Inouye S, Taniai M, Ando S, Usui M, Matuhasi T. Identification of the second major allergen of Japanese cedar pollen. Allergy 1990;45:309-312

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

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[56] - di Felice G, Barletta B, Tinghino R, Pini C. Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens. Int Arch Allergy Immunol 2001;125:280-289

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity

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[57] - Hon SM, Lee BW, Tan HTW, Chew FT. Cloning and expression of an oil palm (Elaeis guineensis Jacq.) pollen allergen showing homology to polygalacturonase. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°924

Background of study: The oil palm (Elaeis guineensis Jacq.) is an important crop in Southeast Asia. Its pollen has been shown to be an important cause of allergy in the tropical regions, with approximately 40% of the atopic population showing skin prick responses to its crude extract [J Allergy Clin Immunol 95 (1 pt 2): 261]. Method used and results obtained: Here we report the isolation and cloning on an oil palm pollen allergen that show some homology to the Cry j 2, the second major Japanese cedar pollen allergen. This clone was selected and cloned from an oil palm pollen cDNA library, and further full-length was obtained via the RACE (Rapid Amplification of cDNA Ends) technique. It has a complete sequence of 1513 bp, with an open reading frame of 399 amino acids and six potential glycosylation sites. It has a derived molecular weight of approximately 42kDa. Alignment of its deduced amino acid sequences show approximately 34% homology to Cry j 2, the second major Cryptomeria japonica (Japanese cedar) pollen, 31% with Cha o 2, the Japanese cypress pollen, and 30.4% with Jun a 2, the major pollen allergen of Juniperus ashei. A fusion protein induced by IPTG from the expression host consisting pET32a(+) vector showed a distinct thick band at approximately 60kDa. The recombinant protein was then purified by affinity chromatography using pET His.Tag system. The purified protein was tested via immuno-dot blot against 5 sera obtained from individuals who were skin prick sensitized to oil palm pollen. A total of 1 ug of the protein were dotted on nitrocellulose membrane and incubated overnight with the sera at 4°C. The protein showed positive IgE binding reaction towards all 5 sera. No IgE binding was observed in the negative control sera. Conclusion: In conclusion, this polygalacturonase-like pollen protein is likely a new allergen identified from the oil palm pollen besides the allergens identified earlier from our previous studies; a profilin and pectin esterase-like protein (J Allergy Clin Immunol 105 (1 pt 2): 333). These recombinant proteins may be useful for future diagnostic and therapeutic purposes.

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[58] - Futamura N, Kusunoki Y, Mukai Y, Shinohara K. Characterization of Genes for a Pollen Allergen, Cry j 2, of Cryptomeria japonica. Int Arch Allergy Immunol 2007;143:59-68

BACKGROUND: Cry j 2 is one of the major pollen allergens of Cryptomeria japonica. The polymorphism of Cry j 2 isoforms and the conservation of the structure of Cry j 2 in coniferous species remain to be analyzed . METHODS: A cDNA library derived from the pollen of C. japonica was screened using a fragment of Cry j 2 cDNA. Restriction fragment length polymorphism analysis was performed to examine the diversity of Cry j 2 genes. The promoters of Cry j 2 genes were isolated with a commercially available cloning kit. Clonal variations in the expression of Cry j 2 in pollen were examined by RNA gel blot analysis, and the conservation of the structure of the Cry j 2 gene in coniferous species was evaluated by DNA gel blot analysis . RESULTS: We isolated three cDNA clones encoding novel isoforms of Cry j 2. We also sequenced a total of 16 promoter regions from 10 specimens. The sequences of promoter regions of Cry j 2 genes were highly divergent. The amount of Cry j 2 mRNA also varied considerably. The Cry j 2 gene was found to be conserved among species belonging to Taxodiaceae and Cupressaceae but to vary between Taxodiaceae and Pinaceae . CONCLUSIONS: The coding and promoter regions of Cry j 2 genes contain large numbers of polymorphisms. Our analysis revealed large variations in the expression of Cry j 2 at the transcriptional level, and we suggest that conserved homologs of Cry j 2 confer cross-allergenicity among Taxodiaceae and Cupressaceae.

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[59] - Hashimoto M, Nigi H, Sakaguchi M, Inouye S, Imaoka K, Miyazawa H, et al. Sensitivity to two major allergens (Cry j I and Cry j II) in patients with Japanese cedar (Cryptomeria japonica) pollinosis. Clin Exp Allergy 1995;25:848-852

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 histamine 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 o f 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

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[60] - Krebitz M, Wagner B, Ferreira F, Peterbauer C, Campillo N, Witty M, et al. Plant-based Heterologous Expression of Mal d 2, a Thaumatin-like Protein and Allergen of Apple (Malus domestica), and its Characterization as an Antifungal Protein. J Mol Biol 2003;329:721-730

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.

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[61] - Soman KV, Midoro-Horiuti T, Ferreon JC, Goldblum RM, Brooks EG, Kurosky A, et al. Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3. Biophys J 2000;79:1601-1609

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

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[62] - Lahoz C, Cortegano I, Minguez A, Del Pozo V, Civantos E, Cardaba B, et al. Cup a 3, Major Allergen From Cupressus Arizonica: Cloning and Expression in Pichia Pastoris. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°397

The Cupressaceae family is a relevant source of pollen allergens in the Mediterranean area responsible for frequent winter respiratory allergies. We have previously cloned the major allergen from Cupressus arizonica (Cup a 1): Clin. Exp. Allergy 2000;30:1750-1758, and now we describe the cloning and expression of an important allergen from this family: Cup a 3 which is reactive with more than 90% of cupressaceae patients, being considered as a major antigen with similar reactivity to Cup a1. The theoretical molecular weight for the cloned protein is 21 kDa, the calculated pI is 4.82 and several phosphorylation sites are found in the amino acid sequence. The nucleotides sequence NCBI Nucleotide Database (AJ294411, Cup a 3) has 96% homology with mountain cedar allergen Jun a 3, 93% with Vitis vinifera a thaumatin-like protein and 89% with Atriplex nummularia an osmotin-like protein. This allergen may be included in Group 5 of pathogenesis-related proteins. Recombinant Cup a 3 has been cloned in pBluescript and expressed in E. coli as a fusion protein with GST but the yield was poor. To improve it, Cup a 3 has been expressed in Pichia pastoris system using pPCIZA plasmid. A cDNA encoding Cup a 3, fused to a - factor peptide using the pPCIZA vector has been inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein is secreted into the extracellular medium. The IgE binding properties of the recombinant protein produced in yeast have been tested in immunoblot assays using sera from allergic patients. The excellent production of recombinant Cup a 3 in yeast may contribute to the standardization of the allergens used for diagnosis and immunotherapy. The nucleotide sequence is: 1 gtgaagtttg atataaagaa ccagtgcggg tacactgtct gggcagcggg gttgcccgga 61 ggagggaagg agtttgacca ggggcagaca tggacggtta atttggcggc gggcacagcg 121 tcggcaaggt tctggggacg aacgggctgc actttcgatg cgagcgggaa aggaagctgc 181 cggagcggtg actgcggcgg gcaactgagc tgcacagtct ccggagcagt tcccgcaacg 241 ctggcagagt acacgcagag cgaccaggac tactacgacg tctccctcgt cgatggcttc 301 aacattcctc ttgccatcaa cccaaccaat acaaaatgca ctgcccctgc ctgcaaggct 361 gacattaatg cagtgtgccc ttccgagttg aaggttgatg gcggatgcaa cagtgcctgc 421 aatgtcttac aaactgatca gtattgctgc agaaatgcgt atgttaataa ctgccctgcc 481 acgaattact ccaagatatt caagaaccag tgccctcagg cttatagcta tgctaaggat 541 gacactgcca ctttcgcttg cgcctctggt accgactaca gtattgtatt ctgcccc.

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[63] - Midoro-Horiuti T, Goldblum RM, Kurosky A, Wood TG, Brooks EG. Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen. J Immunol 2000;164:2188-2192

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.

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[64] - Fujimura T, Futamura N, Midoro-Horiuti T, Togawa A, Goldblum RM, Yasueda H, et al. Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen. Allergy 2007;62:547-563

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.

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[65] - Togawa A, Panzani RC, Garza M, Goldblum RM, Midoro-Horiuti T. Isolation and Characterization of the Major Allergen From Italian Cypress (Cupressus sempervirens) Pollen. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°1099

Rationale The prevalence of seasonal allergic diseases of the upper airways is high in many industrialized countries and increasing. The family Cupressaceae is an important cause of pollinosis, particularly in Europe. Based on our previous studies of mountain cedar pollen, we hypothesized that the Italian cypress pollen would contain an allergenic, pathogenesis-related group 5 (PR-5) protein. We therefore sought to clone a cDNA from that pollen, based on nucleotide homology to Jun a 3 of mountain cedar. Method s : mRNA was purified from Italian cypress pollen. cDNA was synthesized and portion of a PR-5 message was amplified by PCR and extended by RACE methods. Recombinant Cup s 3 was expressed as a fusion protein with maltose binding protein. The allergenicity of Cup s 3 was established using an ELISA inhibition assay, based on crude extracts of Italian cedar pollen, the recombinant fusion protein of Cup s 3 and sera from France patients allergic to Italian cypress. Result s : Three isotypes, Cup s 3.1, Cup s 3.2 and Cup s 3.3. were identified. These had 95.5% amino acid sequence identity to Jun a 3. Recombinant Cup s 3.2 inhibited the binding of IgE from the majority of sera from patients to an extract of Italian cypress. Conclusions : Cup s 3 is a major allergen of Italian cypress. Anticipated variations in the content of Cup s 3 in pollen from different region/trees should be considered in the preparation of extracts for diagnostic and therapeutic use.

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[66] - Fujimura T, Futamura N, Midoro-Horiuti T, Togawa A, Goldblum RM, Yasueda H, et al. Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen. Allergy 2007;62:547-563

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.

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[67] - Futamura N, Mukai Y, Sakaguchi M, Yasueda H, Inouye S, Midoro-Horiuti T, et al. Isolation and characterization of cDNAs that encode homologs of a pathogenesis-related protein allergen from Cryptomeria japonica. Biosci Biotechnol Biochem 2002;66:2495-2500

Many plant pathogenesis-related (PR) proteins are allergenic. We isolated three cDNAs, Cry j 3.1, Cry j 3.2, and Cry j 3.3, that encoded homologs of Jun a 3, a PR protein allergen in Juniperus ashei, from a cDNA library derived from the pollen of Cryptomeria japonica. The predicted amino acid sequences encoded by the three cDNAs were more than 85% identical to each other and about 57% identical to the sequence of Jun a 3. The Cry j 3 genes seemed to form a small multigene family in the genome of C. japonica. Expression of Cry j 3 was strong in roots and in female and male strobili; expression was weaker in cotyledons, leaves, stems, and pollen grains.

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[68] - Togawa A, Panzani RC, Garza MA, Kishikawa R, Goldblum RM, Midoro-Horiuti T. Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity. Ann Allergy Asthma Immunol 2006;97:336-342

BACKGROUND: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. OBJECTIVE: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. METHODS: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. RESULTS: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. CONCLUSION: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.

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[69] - Fujimura T, Futamura N, Midoro-Horiuti T, Togawa A, Goldblum RM, Yasueda H, et al. Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen. Allergy 2007;62:547-563

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.

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[70] - Barderas R, Villalba M, Rodriguez R. Recombinant expression, purification and cross-reactivity of chenopod profilin: rChe a 2 as a good marker for profilin sensitization. Biol Chem 2004;385:731-737

Chenopod pollen is one of the major sources of allergens in some locations in the US, southern Europe and desert countries, and pollen profilin (Che a 2) is a major allergen. Recombinant Che a 2 (rChe a 2) has been produced in Escherichia coil cells with a final yield of 25 mg/l of cell culture. The expressed protein was isolated and structurally characterized by means of mass spectrometry, Edman degradation and circular dichroism. rChe a 2 displayed a molecular mass of 13 959 Da, which agrees with that of the amino acid sequence. The N-terminal amino acid sequence indicated the correct processing of the recombinant product. The immunological analysis of rChe a 2 showed IgG- and IgE-binding capabilities equivalent to those of its natural counterpart, Che a 2, isolated from the pollen. Inhibition experiments showed high cross-reactivity degrees with different allergenic sources. Inhibition degrees of >95% and >80% were obtained for chenopod profilin and, respectively, latex and pollen extracts, whereas 10-95% of inhibition was observed for different plant-derived foods. Due to its close relation to other allergenic profilins from pollens, plant-derived foods and latex, rChe a 2 could be a useful tool in clinical trials to detect profilin-allergic patients and perhaps, depending on its clinical relevance, in specific immunotherapy of these hypersensitive individuals.

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[71] - Tinghino R, Barletta B, Palumbo S, Afferni C, Iacovacci P, Mari A, et al. Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen. J Allergy Clin Immunol 1998;101:772-777

BACKGROUND: Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries. OBJECTIVE: We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupressaceae family. METHODS: Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector. IgE screening of the library was performed with a pool of sera from subjects allergic to Cupressaceae. A recombinant 6xHis-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. It was studied further by immunoblotting inhibition with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae. The role of protein-bound calcium on the allergen's IgE-binding capacity was tested in a plaque assay in the presence or absence of EGTA. RESULTS: A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated. The deduced amino acid sequence contained four typical Ca2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2. Immunoblotting inhibition revealed that J. oxycedrus, J. ashei, Cupressus arizonica, C. sempervirens, Parietaria judaica, Olea europaea, and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations. CONCLUSION: rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen. These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered.

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[72] - Ledesma A, Villalba M, Vivanco F, Rodríguez R. Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8-like proteins in different pollens. Allergy 2002;57:40-43

In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli. We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8). Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen. Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens. Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other. Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.

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[73] - Kawamoto S, Fujimura T, Nishida M, Tanaka T, Aki T, Masubuchi M, et al. Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family. Clin Exp Allergy 2002;32:1064-1070

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.

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[74] - Fujimura T, Shigeta S, Suwa T, Kawamoto S, Aki T, Masubuchi M, et al. 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. Clin Exp Allergy 2005;35:234-243

Summary 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.

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[75] - Fujimura T, Shigeta S, Suwa T, Kawamoto S, Aki T, Masubuchi M, et al. 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. Clin Exp Allergy 2005;35:234-243

Summary 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.

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[76] - Bistoni O, Emiliani C, Agea E, Russano AM, Mencarelli S, Orlacchio A, et al. Biochemical and Immunological Characterization of Pollen-Derived ß-Galactosidase Reveals a New Cross-Reactive Class of Allergens among Mediterranean Trees. Int Arch Allergy Immunol 2005;136:123-133

BACKGROUND: The most potent allergens in the Spermatophytae family exhibit significant homology with enzymes. Some of these are though to be involved in pectin metabolism, recognition of compatible stigma and delivery of sperm cells to the ovule . OBJECTIVE: To test if glycohydrolase activities from some Mediterranean tree pollens could act as allergens in sensitized hosts . METHODS: Freshly collected Cupressus and Olea pollens were investigated for their glycohydrolase activities by means of synthetic fluorogenic substrates and isoenzymes characterized by DEAE-cellulose ion-exchange chromatography. Binding of specific IgE was investigated by immunoblotting in 30 tree-sensitive subjects, as well as in 20 atopic non-tree-sensitive and 15 healthy controls. The enzymes were also adopted to stimulate proliferation of allergen-specific T cell clones. Finally, they were tested in vivo in a cutaneous immediate wheal and flare reaction . RESULTS: beta-Galactosidase (beta-GAL) is present with different isoenzymatic patterns on both pollen extracts, could be recognized by circulating IgE, as well as immunoprecipitated by sera from allergic subjects. The enzyme could stimulate the proliferation of T cells from allergic subjects, and favor the emergence of CD4+ T cell clones with specific in vitro reactivity to beta-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects . CONCLUSIONS: Despite different isoenzymatic patterns, Olea-derived beta-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.

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[77] - Suarez-Cervera M, Vega-Maray A, Asturias J, Castells T, Rodriguez-Rajo J, Moreno-Grau S, et al. Immunocytochemical localization of a lipid transfer protein in Cupressus arizonica pollen grains. Allergy 2008;63(suppl. 88):654

Background: Lipid transfer proteins (LTPs) are relevant allergens since they have been involved in cross-reactivities among pollen and fruit allergenic sources, and therefore they have been named panallergens. It is assumed that LTPs from different species present diverse homologies, such as the peach LTP, Pru p 3, which has sequence similarities with Platanus and Artemisia pollen grains from plants which are botanically distant from the Rosaceae family. The aim of this study is to localize the cypress LTP in the pollen grains of Cupressus arizonica using a combination of transmission electron microscopy, immunocytochemical techniques, and rabbit specific antiserum against peach LTP. Methods: Observations were made in mature and hydrated C. arizonica pollen grains from Barcelona, Spain. Specimens were fixed using freezing protocols and ultrathin sections were incubated with anti-Pru p 3 rabbit polyclonal antibodies. Results: Pru p 3-like LTP was localized in the cytoplasm and walls of C. arizonica pollen grains in mature and hydratedactivated stages showing that a cross-reaction exists between peach and Cupressus LTPs. Abundant labelling was observed in the dormant cytoplasm and nucleus. At the beginning of activation, the labelling was found during the development of the membrane systems. In the activated cytoplasm, the gold particles were associated with lipid globules, vacuoles and the Golgi complex. The cytoplasm localization of LTPs in C. arizonica, associated with lipid globules, indicates an important role in the lipid metabolism. This role is particularly relevant at the level of the Golgi complex in the secretory cell pathways. No labelling was observed in control sections incubated with preimmune rabbit serum. Conclusions: This observation of the antigenic cross-reactivity between Pru p 3 and the Cupressus LTP makes this pollen a strong candidate to be considered in the cross-reaction between Rosaceae fruits and pollen grains. Therefore, future studies are needed to characterize the Cupressus LTP and evaluate its clinical significance.

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[78] - Shahali Y, Sutra J, Desvaux F, Peltre G, Calleja M, Charpin D et al. Identification and characterisation of pollen allergens in two cypress species by IEF, SDS-PAGE and immunoblotting. Allergy 2009;64(Suppl. 90):376

Background: Allergy to Cupressaceae is becoming a serious health problem in some regions worldwide commonly inducing symptoms of hay fever, allergic conjunctivitis, hacking cough and asthma in sensitized individuals. In this study we aimed to identify and characterize pollen allergens of Mediterranean cypress (C. sempervirens, CS) and Arizona cypress (C. arizonica, CA) representing the major Cupressus spp. growing in France. Methods: To optimize pollen protein extraction, several cypress pollen preparations were analyzed by isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specific IgE-binding to cypress pollen allergens were investigated by immunoblotting using sera of cypress sensitive patients. The optimized pollen extract was finally resolved by two dimensional electrophoresis (2-DE) and assayed with sera of allergic subjects. Results: Our quantitative and qualitative protein studies indicated that pollen extracts obtained by incubating pollen in a solution containing 2% w/v of CHAPS provided a higher amount and wider diversity of proteins than those prepared by extraction in other aqueous solutions (water or 0.01 M PBS). Silver staining of IEF gels detected at least 17 protein bands in CS pollen extracts, including 15 acidic proteins with isoelectric points (pI) ranging from 4.5 to 7 and two bands with approximate pI values of 9 and 10.5. IEF separation profile of CA pollen extracts showed similar acidic bands with a higher protein concentration in the pI region 4.45- 5.1 but three different basic proteins with a pI ranging from 8 to 9.5. SDS-PAGE (8-18%) and immunoblot patterns of CS and CA extracts distinguished IgE-binding proteins with apparent molecular weights of 35, 43, 55, 70 and 90 kD. The 2-DE immunoblotting performed with individual sera revealed an intense immunoreactivity to an acidic allergen (pI 4.5) of about 43 kD and weak IgE responses to various other spots located in a wide range of isoelectric points. Conclusion: Immunoblotting analysis of IEF, SDS-PAGE and 2-DE gels are complementary and useful methods for the characterization and comparison of cypress pollen extracts, expanding the panel of cypress-related allergenic molecules recognition for a more comprehensive allergen-based diagnosis and therapy.

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[79] - Asero R, Jimeno L, Barber D. Prevalence and clinical relevance of hypersensitivity to pollen pan-allergens (polcalcin and Profilin): a SPT study. Allergy 2009;64(Suppl. 90):376-377

Background: Calcium binding proteins (polcalcins) and profilin are cross-reacting pan-allergens that sensitize a minority of pollenallergic patients. Their clinical relevance is still debated. Objective: To assess the clinical relevance of polcalcin and profilin hypersensitivity, detected for the first time by SPT, in a large group of pollen-allergic patients. Methods: 200 pollen-allergic adults (M/F 101/99; mean age 34 years) underwent SPT with 9 pollens present in this geographical area. Hypersensitivity to pan-allergens was detected by SPT with date palm polcalcin and profilin. Three symptomatic periods (from end February to mid May, from end April to mid July, and from mid August to the end of September) were considered as associated with birch and/ or cypress, grass and/or pellitory, and ragweed and/or mugwort allergy, respectively. Results: 16 (8%) patients reacted to date palm polcalcin; 5/5 (100%) co-recognized Phl p 7, the grass polcalcin, in-vitro. Clinically, only 4 (25%) reported symptoms in all 3 seasonal periods. 40 (20%) patients reacted to profilin; only 32 (80%) and 22 (55%) of them reacted to cypress and pellitory pollen, respectively. Clinically, only 4/40 (10%) had symptoms during all 3 seasonal periods. Six patients (3%) were co-sensitized to both polcalcin and profilin. Conclusions: The clinical relevance of hypersensitivity to pollen pan-allergens is often limited; many reactors have symptoms only during the central period, suggesting primary grass sensitization. Profilin reactors often do not co-recognize pellitory and cypress pollen. Invivo component-resolved diagnosis of seasonal respiratory allergies is a promising approach that might lead to reduction of costs and to a prompter definition of cases of multiple pollen reactivity.

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[80] - Weber RW. Patterns of pollen cross-allergenicity. J Allergy Clin Immunol 2003;112:229-239

Knowledge of patterns of pollen cross-reactivity is crucial for diagnostics and especially for formulation of immunotherapy vaccines in times of diminishing availability of pollen extract constituents. As phylogenetic relationships have become better clarified, it becomes apparent that cross-reactivity does reflect taxonomy in the very great majority of cases. Contradictory observations of unexpected cross-reactivity between unrelated plants, sometimes remarkably distant ones, require explanation. There are many proteins, presumably performing vital functions, that are tightly preserved throughout the evolutionary tree from plants to animals, such as profilins, lipid transfer proteins, and pathogenesis-related proteins. These might function as panallergens. The small differences that exist between these ubiquitous proteins explain why these are frequently minor allergens not reacting in the majority of allergic sera. This review summarizes cross-reactivity studies with both crude pollen extracts and purified or recombinant allergenic proteins. The patterns of cross-allergenicity that emerge should be helpful in guiding both diagnostic and therapeutic decisions.

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[81] - Panzani RC, Yasueda H, Shimizu T, Shida T. Cross-reactivity between the pollens of Cupressus sempervirens (common cypress) and of Cryptomeria japonica (Japansese cedar). Ann Allergy 1986;57:26-30

A clinical and immunologic study was performed comparing a group of French patients allergic to the pollens of cypress (Cupressus sempervirens, which belongs to the Cupressaceae family) and a group of Japanese patients allergic to the pollens of Sugi (Cryptomeria japonica, which belongs to the Taxodiaceae family). By skin testing, RAST, and RAST inhibition, clear cross-reactivity was detected between the two pollens. No cross-reactivity was detected between the pollens of Cupressus sempervirens and Sugi and the Pinaceae family. In addition, one can speculate that an antigen in Cupressus sempervirens is cross-reactive with SBP, the major allergen of Sugi, suggesting that there is a closer relationship between the Taxodiaceae family and the Cupressaceae family than between these two families and the other families of the gymnosperms. This finding throws new light on the taxonomy of the gymnosperms.

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[82] - Nakamura Y, Takagi S, Suzuki M, Ito H, Murakami S, Ohta N. Survival of memory T cells specific for Japanese cypress pollen allergen is maintained by cross-stimulation of putative pectate lyases from other plants. Allergy 2001;56:385-392

In view of recent studies on the mechanisms of the survival of peripheral memory T cells, we tested the biologic role of pectate lyase, a pectin-degrading enzyme, as the cross-reactive antigen required for the recurring survival signals for human T cells specific for Cha o 1, a pollen allergen molecule of the Japanese cypress. We determined a 16-mer epitope peptide for the T-cell clone, and prepared synthetic oligopeptides of homologous regions in putative pectate lyase of other plants. Of these homologous peptides, ZePel (Zinnia elegans), ban 17 (banana), and Amb a 1.1 (short ragweed) induced strong proliferative responses of the Cha o 1-specific T-cell clone in vitro. In addition, suboptimal doses of peptide homologs derived from banana and short ragweed enhanced the survival potency of this T-cell clone without detectable proliferative responses to the peptides. When there was no antigen stimulation, the T-cell clone decreased in viable cell number and lost antigen-specific proliferation activity on day 6 during in vitro incubation. On the other hand, T-cell clones incubated with these survival-inducing peptides maintained proliferative activity to Cha o 1 even on day 9. Serum derived from the donor patient did not contain detectable levels of IgE specific to banana or short ragweed by CAP-RAST. These results show that human T cells specific for pollen allergen seem to use cross-reactive pectate lyase peptides to deliver survival signals even in the absence of pollen allergen, and memory T cells maintained in such a manner might be functioning at the onset of allergic pollinosis, although pollen allergens are seasonal.

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[83] - Sone T, Dairiki K, Morikubo K, Shimizu K, Tsunoo H, Mori T, et al. 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. Clin Exp Allergy 2005;35:664-671

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.

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[84] - Bohle B. The impact of pollen-related food allergens on pollen allergy. Allergy 2007;62:3-10

Patients with birch pollen allergy frequently develop hypersensitivity reactions to certain foods, e.g. apples, celery, carrots and hazelnuts. These reactions are mainly caused by IgE-antibodies specific for the major birch pollen allergen, Bet v 1, which cross-react with homologous proteins in these foods. Analyzing the T-cell response to Bet v 1-related food allergens revealed that these dietary proteins contain several distinct T-cell epitopes and activate Bet v 1-specific T cells to proliferate and produce cytokines. Several of these cross-reactive T-cell epitopes were not destroyed by simulated gastrointestinal digestion of food allergens and stimulated Bet v 1-specific T cells despite nonreactivity with IgE antibodies. Similarly, cooked food allergens did not elicit IgE-mediated symptoms (oral allergy syndromes) but caused T-cell-mediated late-phase reactions (deterioration of atopic eczema) in birch pollen-allergic patients with atopic dermatitis because thermal processing affected their conformational structure and not the primary amino acid sequence. Thus, T-cell cross-reactivity between Bet v 1 and related food allergens occurs independently of IgE-cross-reactivity in vitro and in vivo. We speculate that symptom-free consumption of pollen-related food allergens may have implications for the pollen-specific immune response of allergic individuals.

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[85] - Enomoto T, Onishi S, Sogo H, Dake Y, Ikeda H, Funakoshi H, et al. Japanese cedar pollen in floating indoor house dust after a pollinating season. Allergol Int 2004;53:279-285

Background: Approximately 16.2% of the Japanese population suffers from pollinosis. One of the forms of management is self-care (preventive care), which can be categorized as 'indoor' and 'outdoor'. Outdoor self-care is usually emphasized, but indoor self-care is also important. Considerable pollen is found in indoor dust and this is thought to be one of the factors that worsens pollinosis and enables it to persistent for a long time, even after the pollinating period has finished. Taking this into consideration, we investigated the dynamic state of indoor pollen. Methods: Floating indoor house dust was collected in Petri dishes. The amount of pollen in the house dust samples collected was measured using an LCD laboratory highly sensitive Cry j1 assay kit. Results: The results showed that, indoors, a lot of Japanese cedar pollen (JCP) was found on the floor (tatami mats, carpets), sofas and curtains. The number of JCP in living rooms peaked in April after the pollinating period and decreased gradually; however, JCP was still found indoors, even as late as the following February. Floating JCP in the house was one-tenth of the JCP levels on the floor. Floating JCP increased on days with low humidity. Air conditioning temporarily increased levels of floating JCP in houses with an air conditioner, but the level of floating JCP decreased rapidly compared with the level of that in houses without an air conditioner. Nasal signs and symptoms disappeared completely at a level of 30 floating pollen counts/day per Petri dish. Conclusion: Considerable JCP was found floating indoors with house dust after a pollinating season.

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[86] - Kondo Y, Urisu A, Tokuda R. Identification and Characterization of the Allergens in the Tomato Fruit by Immunoblotting. Int Arch Allergy Immunol 2001;126:294-299

Background: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Methods: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients' sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. Results: Four proteins binding with IgE from more than half of the OAS patients' sera were determined to be polygalacturonase 2A (PG2A), -fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, -fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. Conclusion: The concentrations of 3 of 4 tomato allergens increased during ripening.

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[87] - Tinghino R, Hayek B, Barletta B, Puggioni E, Iacovacci P, Butteroni C, et al. Ca-binding allergens: cross-reactivity between molecules with 2 (rAln g4) and 4 (rJun o2) EF-hand domains. J Allergy Clin Immunol 2000;105(1 part 2):S332

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[88] - Pham NH, Baldo BA. Allergenic relationship between taxonomically diverse pollens. Clin Exp Allergy 1995;25:599-606

Skin tests and tests for IgE antibodies show that subjects are usually sensitive to a number of different pollens, frequently from taxonomically diverse species which are assumed to be allergenically non-crossreactive. This suggests that the presence of IgE antibody-reactivity to an individual pollen may not necessarily have resulted from contact with that pollen or even with a taxonomically closely related species. OBJECTIVE: Since this has important consequences for allergen avoidance and desensitization of patients, we attempted to define allergenic relationships between diverse pollen species. METHODS: Sera from subjects were examined in direct IgE antibody binding experiments and by quantitative inhibition, protein blotting and adsorption and elution studies. RESULTS: Sera from subjects diagnosed as allergic to white cypress pine, Italian cypress, ryegrass or birch pollen were shown to have IgE antibodies that reacted with pollens from these four species and from cocksfoot, couch grass, lamb's quarter, wall pellitory, olive, plantain and ragweed. These reactions were confirmed in protein blotting and adsorption and elution studies where numerous IgE-binding bands were detected in all 11 different pollen extracts with sera from each of the different allergic categories. Further evidence of allergenic (i.e. IgE-binding crossreactivity between the different pollens was provided by inhibition studies in which clear-cut inhibitions of IgE binding to the different pollen allergen discs were obtained with comparable amounts of the different pollen extracts. CONCLUSION: We conclude that the presence of pollen reactive IgE antibodies may not necessarily be a true reflection of sensitizing pollen species.

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[89] - Gonzalez EM, Villalba M, Rodriguez R. Allergenic cross-reactivity of olive pollen. Allergy 2000;55:658-663

BACKGROUND: Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. METHODS: We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS-PAGE and inhibition analysis of this binding. RESULTS: A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. CONCLUSIONS: Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.

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[90] - Bar Dayan Y, Keynan N, Waisel Y, Pick AI, Tamir R. Podocarpus gracilior and Callitris verrucosa--newly identified allergens that crossreact with Cupressus sempervirens. Clin Exp Allergy 1995;25:456-460

Thirty-six symptomatic patients, with positive skin reactions to Cupressus sempervirens pollen extract were skin-tested with pollen extracts of Podocarpus gracilior and Callitris verrucosa, of these 17 (47%) had positive responses to P. gracilior, nine (25%) to C. verrucosa, and six (17%) to both. None of the non-atopic healthy controls had positive reactions to either of the extracts. Radioallergosorbent test (RAST)-inhibition studies were performed with pooled sera from three patients. Fifty per cent inhibition was obtained with 11 micrograms protein of C. sempervirens, 54 micrograms of P. gracilior, and 71 micrograms of C. verrucosa; however, when pollen extract of Olea europaea, an unrelated allergen, was tested, 265 micrograms protein were needed to obtain 50% inhibition. One-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pollen extracts from the three species revealed that they had several very similar protein bands. Using Western blot analysis, several closely related IgE binding proteins were identified in the three species. It was concluded that the pollen grains of P. gracilior and of C. verrucosa are potentially allergenic. The presence of common allergenic proteins indicate partial crossreactivity with C. sempervirens.

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[91] - Pham NH, Baldo BA, Bass DJ. Cypress pollen allergy. Identification of allergens and crossreactivity between divergent species. Clin Exp Allergy 1994;24:558-565

Studies employing sera from 34 subjects allergic to white cypress pine (Callitris glaucophylla) pollen identified 18 IgE antibody-binding components in the pollen of this species, five of which (MWs approximately 94, 68, 64, 43 and 34 kDa) were recognized by all of the sera. Protein blotting and quantitative inhibition studies revealed clear cross-reactivity between C. glaucophylla and Cupressus sempervirens pollen proteins and striking similarities in the IgE recognition band patterns of the two pollens. Inhibition experiments with other pollen extracts revealed that sera from C. glaucophylla pollen-allergic subjects can be divided into two groups--those inhibited only by extracts from the two Cupressaceae pollens and those inhibited both by these pollen proteins and by pollen extracts from other species. Most of the crossreactions in the latter group cannot be explained on the basis of taxonomic relationships or separate sensitizations. As with previous studies on birch and olive pollens, we conclude that pollen allergenic crossreactivity is much more wide-ranging than generally believed.

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[92] - Bistoni O, Emiliani C, Agea E, Russano AM, Mencarelli S, Orlacchio A, et al. Biochemical and Immunological Characterization of Pollen-Derived ß-Galactosidase Reveals a New Cross-Reactive Class of Allergens among Mediterranean Trees. Int Arch Allergy Immunol 2005;136:123-133

BACKGROUND: The most potent allergens in the Spermatophytae family exhibit significant homology with enzymes. Some of these are though to be involved in pectin metabolism, recognition of compatible stigma and delivery of sperm cells to the ovule . OBJECTIVE: To test if glycohydrolase activities from some Mediterranean tree pollens could act as allergens in sensitized hosts . METHODS: Freshly collected Cupressus and Olea pollens were investigated for their glycohydrolase activities by means of synthetic fluorogenic substrates and isoenzymes characterized by DEAE-cellulose ion-exchange chromatography. Binding of specific IgE was investigated by immunoblotting in 30 tree-sensitive subjects, as well as in 20 atopic non-tree-sensitive and 15 healthy controls. The enzymes were also adopted to stimulate proliferation of allergen-specific T cell clones. Finally, they were tested in vivo in a cutaneous immediate wheal and flare reaction . RESULTS: beta-Galactosidase (beta-GAL) is present with different isoenzymatic patterns on both pollen extracts, could be recognized by circulating IgE, as well as immunoprecipitated by sera from allergic subjects. The enzyme could stimulate the proliferation of T cells from allergic subjects, and favor the emergence of CD4+ T cell clones with specific in vitro reactivity to beta-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects . CONCLUSIONS: Despite different isoenzymatic patterns, Olea-derived beta-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.

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[93] - Ledesma A, Villalba M, Rodriguez R. Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity. FEBS Lett 2000;466:192-196

A novel allergenic member of the family of Ca(2+)-binding proteins has been cloned from olive tree pollen. The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF-hand sequence motifs. The encoded protein was overproduced in Escherichia coli and purified. The protein (18: omitted:795 Da), which binds Ca(2+) and IgE antibodies from patients allergic to olive pollen, undergoes Ca(2+)-dependent conformational changes. It is retained on a phenyl-Sepharose column, which indicates the existence of regulatory EF-hand domains. This fact suggests its involvement in Ca(2+)-dependent signal transduction events of the pollen grain. This allergen could be considered as a member of a new subfamily of EF-hand Ca(2+)-binding proteins since it displays a low amino acid sequence similarity with the so far known proteins.

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[94] - Kondo Y, Tokuda R, Urisu A, Matsuda T. Assessment of cross-reactivity between Japanese cedar (Cryptomeria japonica) pollen and tomato fruit extracts by RAST inhibition and immunoblot inhibition. Clin Exp Allergy 2002;32:590-594

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. Method s : 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. Result s : 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

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[95] - Kawamoto H, Yamagata M, Nakashima H, Kambe M, Kuraoka T. [A case of tomato juice-induced oral allergy syndrome in which dyspnea onset occurred during the season of Japanese cedar pollen dispersion]. Nihon Kokyuki Gakkai Zasshi 2003;41:397-401

A 42-year-old-man with a history of Japanese cedar pollinosis repeatedly visited the emergency clinic due to dyspnea during the season of Japanese cedar pollen dispersion. Before each onset of this symptom, he had always drunk tomato juice. Swelling of the oral and nasal mucosa, and congestion of the bulbar conjuctiva was observed. No audible wheezing was present. His pulmonary function test results were normal (FEV 1.0 = 4.02 L, %FEV 1.0-124%, negative reversible test). The CAP RAST scores were 4 for tomatoes and 3 for Japanese cedar pollen. A result was obtained in a challenge test using tomato juice. Since tomato juice was involved in the development of the symptoms, a diagnosis of oral allergy syndrome induced by tomato juice was made. When tomato juice consumption was avoided, no symptoms developed. A common antigenicity was found between tomatoes and Japanese cedar pollen. This may be associated with the development of this allergy during the dispersion season of Japanese cedar pollen. The dyspnea may have reflected a feeling of pharyngeal narrowing which is a symptom of oral allergy syndrome. The possibility of oral allergy syndrome as the chief complaint should be considered also in patients with dyspnea. This is the first reported case of oral allergy syndrome induced by tomato juice.

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[96] - Bonds RS, Tiwari R, Ning B, Czerwinski EW, Goldblum RG, Midoro-Horiuti T. Cross Reactivity in Mountain Cedar-Tomato Oral Allergy Syndrome. J Allergy Clin Immunol 2007;119(1 suppl):S113

RATIONALE: Oral allergy syndrome (OAS) is recognized in up to 50% of pollen-allergic adults. However, the mechanisms of cross-recognition are not well established. We describe here a mountain cedar-tomato OAS and propose a molecular basis for cross-reactivity. METHODS: Eleven patients with tomato OAS and cedar pollen hypersensitivity were identified by history and skin testing. A role for Jun a 1, the dominant allergen of mountain cedar, was evaluated by ImmunoCap inhibition. A panel of monoclonal antibodies (mAbs) to Jun a 1 was screened for reactivity with tomato crude extract (CE) by ELISA. Strongly reacting mAbs were tested by Western blotting and immunoprecipitation, followed by protein idenfification by mass spectrometry. RESULTS: Jun a 1 inhibited 38% of patient IgE binding to tomato ImmunoCaps. MAbs that reacted with partially heat sensitive (conformational) and heat resistant (linear) epitopes of Jun a 1 cross-reacted with tomato CE. Western blot analysis suggested that mAbs bound to several tomato proteins, including some known allergens. Immunoprecipitation and mass spectrometry indicated that the most striking interaction of several mAbs was with tomato pectin methylesterase. Computer overlays of the crystal structures of Jun a 1 and tomato pectin methylesterase demonstrated extensive structural homology, despite a very low degree of sequence identity. CONCLUSIONS: Our findings suggest extensive cross-reactivity between Jun a 1, the major allergen of mountain cedar, and tomato allergens, in particular pectin methylesterase. Careful analysis of the epitopes recognized by IgE in patients with cedar-tomato OAS may allow us to define patterns that predict local and systemic reactions to tomato.

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[97] - Sharma GS, Midoro-Horiuti T, van Bavel JH, Goldblum RM. Oral allergy syndrome to tomato, banana, and apple in mountain cedar pollinosis. ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° 59

Mountain cedar (Juniperus ashei, MC, Cupressaceae) pollen is a major cause of seasonal hypersensitivity in the central US. Some patients with hypersensitivity to other pollen allergens have oral allergy syndrome (OAS), an IgE-mediated reaction of the lips, mouth and pharynx after eating native fruits and vegetables. OAS is thought to be due to IgE anti-pollen antibodies that cross-react with the food allergens. OAS has not been reported in MC pollinosis, but OAS to tomatoes has been described in a few patients with Japanese cedar pollinosis. We performed a mail-out / telephone survey of 800 mountain cedar-sensitive patients in the Austin, TX area. After telephone screening, 28 patients were interviewed, skin tested and had serum collected for serological testing. Of the 28 cedar sensitive patients with suspected food allergies, 15 had clinical manifestations of oral allergy syndrome. Eleven of the 28 subjects had positive skin tests to tomato and 6 of these patients were also positive to banana. Subjects with oral allergy syndrome to tomatoes and bananas tended to have stronger cutaneous and in vitro reactivity to cedar pollen. The intensities of the tomato and banana reactivities were correlated with the intensity of cedar reactivity. Sera from the 11 cedar-sensitive patients with positive skin tests to tomato were tested for IgE antibodies to these fruits by ImmunoCAP. ImmunoCAP inhibition assays with cedar and tomato extracts were performed on the 7 sera with adequate IgE antibodies to one or more of the fruits. The results of the inhibition experiments demonstrated a strong cross-reactivity between IgE antibodies to cedar pollen and the three fruits. The primary sensitization was to mountain cedar, since absorption with cedar pollen extract strongly inhibited reactivity to each of the fruits, but absorption with tomato extract did not significantly inhibit IgE binding to cedar extract. This is the first report of an OAS in mountain cedar pollinosis patients. Sensitivity to tomato, banana and apple should be considered in cedar-sensitive patients and confirmed if symptoms develop.

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[98] - Ivanciuc O, Mathura V, Midoro-Horiuti T, Braun W, Goldblum RM, Schein CH. Detecting Potential IgE-Reactive Sites on Food Proteins Using a Sequence and Structure Database, SDAP-Food. J Agric Food Chem 2003;51:4830-4837

The high incidence of food allergies, including oral allergy syndrome, represent major considerations when introducing new crops and foods. A new structural database of allergenic proteins, SDAP-Food, http://fermi.utmb.edu/SDAP/, has been developed to aid in predicting the IgE-binding potential of novel food proteins and cross-reactivities among known allergens. The site is designed to facilitate the first steps of a decision tree approach to determine the allergenicity of a given protein, based on the sequence and structural similarity to known allergens and their IgE binding sites. Immunological tests can then be used to confirm the predictions. A hierarchical procedure for identifying potential allergens, using a physical property-based sequence similarity index, has been designed to identify regions that resemble known IgE binding sites. As an example, SDAP tools were used to find food allergen sequences similar to an IgE binding site of the Jun a 3 allergen from mountain cedar pollen. The SDAP sequence similarity search matched the Jun a 3 epitope to regions in several food allergens, including cherry (Pru av 2), apple (Mal d 2) and pepper (Cap a 1), which are, like Jun a 3, members of the plant pathogenesis-related (PR-5) protein family. Homology modeling, using our EXDIS/DIAMOD/FANTOM program suite, indicated a similar surface location and structure for the potential epitope region on all of these allergens. The quantitative approach presented here can be used as part of a screening process for potential allergenicity of recombinant food products.

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[99] - Leone P, Menu-Bouaouiche L, Peumans WJ, Payan F, Barre A, Roussel A, et al. Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7-A. Biochimie 2006;88:45-52

The structure of a thaumatin-like protein from banana (Musa acuminata) fruit, an allergen with antifungal properties, was solved at 1.7-A-resolution, by X-ray crystallography. Though the banana protein exhibits a very similar overall fold as thaumatin it markedly differs from the sweet-tasting protein by the presence of a surface exposed electronegative cleft. Due to the presence of this electronegative cleft, the banana thaumatin-like protein (Ban-TLP) acquires a strong (local) electronegative character that eventually explains the observed antifungal activity. Our structural analysis also revealed the presence of conserved residues of exposed epitopic determinants that are presumably responsible for the allergenic properties of banana fruit towards susceptible individuals, and provided evidence that the Ban-TLP shares some structurally highly conserved IgE-binding epitopes with thaumatin-like proteins from fruits or pollen from other plants. In addition, some overlap was detected between the predicted IgE-binding epitopes of the Ban-TLP and IgE-binding epitopes previously identified in the mountain cedar Jun a 3 TLP aeroallergen. The presence of these common epitopes offers a molecular basis for the cross-reactivity between aeroallergens and fruit allergens.

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[100] - Angus AC, Xiong SQ, Mari A, Wang DY, Chew FT. Identification of a full length IgE-binding thaumatin-like protein from the storage mite, Glycyphagus domesticus. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°611

Background: Thaumatin-like proteins have been demonstrated to elicit significant IgE-mediated allergic reactions and associated pollinosis amongst plant-allergic individuals in the Northern hemisphere. This study reports the isolation and characterization of a new thaumatin-like allergen from the storage mite Glycyphagus domesticus. METHODS: A partial sequence encoding a G.domesticus thaumatin-like protein was screened from a directionally-cloned cDNA library, after which the full length cDNA clone was obtained via 5' RACE. Expressed and purified recombinant protein was thereafter assessed for IgE reactivity via immunoblotting studies. RESULTS: The cDNA open reading frame encoded a protein with 213 amino acids and 6 putative glycosylation sites. The full length cDNA sequence shared 66% amino acid identity with Mal d 2, an IgE-reactive thaumatin-like protein. All the conserved cysteines of other thaumatin proteins were also conserved in the G.domesticus homologue, resulting in an overall similarity of its tertiary backbone structures with members of the thaumatin family. The purified recombinant G.domesticus thaumatin-like protein was a 23kDa protein which displayed the ability to bind IgE in 78% of sera (n=136) obtained from Italian pollen-sensitised patients, with a high correlation (r=0.72, p<0.01) existing between positive IgE reactions to this allergen and sensitisation to plant-derived food allergens. IgE binding to this protein however was only observed in 12% of sera (n=120) obtained from mite-sensitized individuals in Singapore. Structural alignment analyses revealed no dramatic disruption of the 3 main IgE binding epitopes detected in another thaumatin-like protein, Jun a 3. CONCLUSION: The significant IgE reactivity reported here indicates that a thaumatin-like protein is an allergen in storage mites, and may warrant further analysis as a potential trans-generic cross-reactive allergen especially amongst individuals exhibiting primary sensitisation to plant allergens.

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[101] - Ishida T, Murai K, Yasuda T, Satou T, Sejima T, Kitamura K. [Oral allergy syndrome in patients with Japanese cedar pollinosis]. Nippon Jibiinkoka Gakkai Kaiho 2000;103:199-205

Patients with pollinosis sometimes complain of oral symptoms (itching and tingling with or without edema of the lips, mouth and tongue) after eating fresh fruits and vegetables. This condition has been termed Oral Allergy Syndrome (OAS). Twenty-three patients with Japanese cedar pollinosis and OAS for fresh fruits and vegetables were included in this study. Their mean age was 31.3 years (range = 5 to 62). The fruits that caused OAS in these patients included melon, apple, peach, and kiwi fruit. Most patients with OAS exhibited hypersensitivity to more than two foods. Specific IgE antibodies to inhaled allergens of mite, Japanese cedar pollen, birch pollen, melon, apple, peach, and kiwi were evaluated using the Pharmacia CAP system. Eleven of the 16 subjects with specific IgE antibodies for birch pollen, did not suffer symptoms during the birch and alder pollen season. In subjects with specific IgE antibodies for fruits, 13 out of 20 patients showed specific IgE antibodies for apple, and 8 out of 9 patients with OAS for apples were also positive for specific IgE antibodies for apples. On the other hand, 17 patients had no specific IgE antibodies for melon, and only two patients and one patient showed specific IgE antibodies for kiwi fruit and peach, respectively. These results suggest that the evaluation of specific IgE antibodies to birch pollen and apple may be useful for diagnosing OAS in patients with Japanese cedar pollinosis.

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[102] - Arai Y, Ogawa C, Ohtomo M, Sano Y, Ito K. [Food and food additives hypersensitivity in adult asthmatics. II. Oral allergy syndrome in adult asthmatic with or without Japanese cedar hay fever]. Arerugi 1998;47:715-719

OBJECTS: The aim of this study was to investigate whether oral allergy syndrome (OAS) in Japan has a particular association with Japanese cedar (JC) hay fever and which kinds of food allergen cause OAS. SUBJECTS AND METHOD: The questionnaire was answered by 463 adult asthmatics. Each patient was submitted to skin scratch tests with fresh foods and commercial food extracts. RESULTS: Of the 463 patients 45 (9.7%) were diagnosed as OAS. The foods, which most often provoked a reaction, were in order of frequency, melon, kiwi, crab and shrimp. The prevalence of OAS was higher in patients with JC hay fever than without JC hay fever. However, a higher prevalence of OAS was also found in house dust mite antibody positive patients than negative patients. There was no difference in the prevalence of OAS between JC hay fever and house dust mite antibody positive patients. CONCLUSION: It is suggested that OAS has no particular association with JC hay fever. OAS in Japan is associated with different foods from other countries such as Scandinavia where apple is frequently associated with OAS.

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[103] - Fujimura T, Shigeta S, Suwa T, Kawamoto S, Aki T, Masubuchi M, et al. 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. Clin Exp Allergy 2005;35:234-243

Summary 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.

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[104] - Larsson H, Norén M, Lidholm J. Purification and immunological comparison of Cry j 1, Cup a 1 and Cup s 1. Allergy 2009;64(Suppl. 90):505

Background: Pollen from species of the Cupressaceae family are known to be important allergen sources in temperate regions and responsible for many cases of pollinosis. Proteins of the pectate lyase family are among the major Cupressaceae pollen allergens. The purpose of this work was to purify pectate lyases from three important Cupressaceae species (Cryptomeria japonica, Cupressus arizonica and Cupressus sempervirens) and investigate their IgE antibody binding and cross reactivity in a widely used assay system for allergen-specific IgE antibodies. Methods: Cry j 1, Cup a 1 and Cup s 1 were purified from pollen extract by affinity chromatography, using an a-Cry j 1 monoclonal antibody (mAb) coupled to NHS-activated gel, followed by buffer exchange. The proteins were analysed by silver-stained SDS PAGE and MALDI TOF MS to confirm their purity and identity. Experimental ImmunoCAP tests were prepared with a saturating amount of each allergen component. IgE antibody binding was studied using serum samples from Cupressaceae pollen sensitized subjects from Japan (n=20), southern Europe (n=10) and USA (n=11). All IgE determinations were performed in an ImmunoCAP 250 assay instrument. Results: Purified Cry j 1, Cup a 1 and Cup s 1 showed no detectable contamination by silver-stained SDS PAGE. The identity of each allergen was unambiguously confirmed by MALDI TOF MS. Cry j 1 appeared as a double band in SDS PAGE, suggesting the presence of isoforms, differential glycosylation or partial cleavage. Cup s 1 was obtained at significantly lower yield than Cry j 1 and Cup a 1. Among all serum samples analysed, IgE binding to the purified allergens occurred across the entire measuring range (0-100 kUA/L). Subjects from the three geographical regions differed somewhat in their IgE antibody recognition of the purified allergens. While the European and American groups displayed no discrimination between Cup a 1 and Cup s 1, one third of the Japanese subjects showed clearly higher binding to Cup s 1. Further, the Japanese subjects showed significantly higher IgE binding to Cry j 1 than to Cup a 1 and Cup s 1 whereas the opposite was true for the European and American subjects. Possible reasons for the observations include differences in primary sensitization in relation to shared and different protein determinants among the allergen components studied. Conclusion: In Europe and North America, Cup a 1 and Cup s 1 appear interchangeable as ImmunoCAP reagents.

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[105] - EXPO: Determinacion de la prevalencia de sensibilizacion a polenes mediante tecnicas de diagnostico molecular. ALK-Abello Ed., 2009, 86pp.

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[106] - Barletta B, Tinghino R, Corinti S, Afferni C, Iacovacci P, Mari A, et al. Arizona cypress (Cupressus arizonica) pollen allergens: Identification of cross-reactive periodate-resistant and -sensitive epitopes with monoclonal antibodies. Allergy 1998;53:586-593

Species of the Cupressaceae family are a worldwide cause of respiratory allergies. We used monoclonal antibodies (mAbs) to investigate the presence and the nature of cross-reacting epitopes shared by various components within Cupressus arizonica pollen extract (CaE) or by CaE and pollen extract from C. sempervirens (CsE). mAbs were produced in mice immunized with whole CaE (4A6 and 5E6) or with the major allergen components (2D5). Their reactivity was investigated by ELISA and immunoblotting before and after CaE periodate treatment. Cross-reactivity was evaluated by ELISA inhibition and immunoblotting. mAbs 2D5 and 4A6 recognized periodate-resistant epitopes, whereas the mAb 5E6 reacted with a periodate-sensitive determinant. The former mAbs recognized epitopes present on CaE major allergen and also shared by other components. mAb 5E6 showed a spread reactivity on CaE, with exclusion of the major allergen. When the three mAbs were tested with CsE, a restricted pattern of reactivity to mAbs 2D5 and 4A6 was obtained, whereas mAb 5E6 maintained a spread reactivity. The CaE major allergen is represented by two components recognized by human IgE and sharing common epitopes, as proven by mAbs reactivity. The use of these mAbs demonstrates that cross-reactivity within CaE components and between CaE and CsE is due to the presence of periodate-sensitive as well as -resistant epitopes.

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[107] - Afferni C, Iacovacci P, Barletta B, Di Felice G, Tinghino R, Mari A, et al. Role of carbohydrate moieties in IgE binding to allergenic components of Cupressus arizonica pollen extract. Clin Exp Allergy 1999;29:1087-1094

BACKGROUND: A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens. OBJECTIVE: The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved. METHODS: The reactivity of IgE with C. arizonica extract, before and after periodate-treatment, was evaluated by immunoblotting and ELISA inhibition. The specificity of carbohydrate-reactive IgE was evaluated by ELISA using unrelated glycoproteins with known sugar composition and structure, such as pineapple bromelain, honeybee venom phospholipase A2, and ovalbumin, before and after periodate treatment. RESULTS: When periodate-treated C. arizonica extract was probed after SDS-PAGE and immunoblotting with patients' IgE, no reactivity could be detected. Furthermore, a very poor inhibitory activity of the periodate-treated C. arizonica extract as compared with the untreated sample could be observed in the ELISA inhibition experiments performed using C. arizonica extract as antigen. When phospholipase A2 and bromelain were used as antigens in ELISA, they were recognized by patients' IgE, whereas ovalbumin was negative. Treatment of phospholipase A2 and bromelain with periodate completely abolishes the IgE reactivity. CONCLUSION: A large portion of the IgE reactivity of Cupressaceae-allergic subjects appears to be associated with sugar moieties of C. arizonica extract which appear to be shared by bromelain and phospholipase A2, thus suggesting that the IgE of patients reacting with such epitopes probably react with beta 1 --> 2 xylose, alpha 1 --> 3 fucose and/or alpha 1 --> 6 fucose.

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[108] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[109] - Afferni C, Iacovacci P, Barletta B, Di Felice G, Tinghino R, Mari A, et al. Role of carbohydrate moieties in IgE binding to allergenic components of Cupressus arizonica pollen extract. Clin Exp Allergy 1999;29:1087-1094

BACKGROUND: A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens. OBJECTIVE: The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved. METHODS: The reactivity of IgE with C. arizonica extract, before and after periodate-treatment, was evaluated by immunoblotting and ELISA inhibition. The specificity of carbohydrate-reactive IgE was evaluated by ELISA using unrelated glycoproteins with known sugar composition and structure, such as pineapple bromelain, honeybee venom phospholipase A2, and ovalbumin, before and after periodate treatment. RESULTS: When periodate-treated C. arizonica extract was probed after SDS-PAGE and immunoblotting with patients' IgE, no reactivity could be detected. Furthermore, a very poor inhibitory activity of the periodate-treated C. arizonica extract as compared with the untreated sample could be observed in the ELISA inhibition experiments performed using C. arizonica extract as antigen. When phospholipase A2 and bromelain were used as antigens in ELISA, they were recognized by patients' IgE, whereas ovalbumin was negative. Treatment of phospholipase A2 and bromelain with periodate completely abolishes the IgE reactivity. CONCLUSION: A large portion of the IgE reactivity of Cupressaceae-allergic subjects appears to be associated with sugar moieties of C. arizonica extract which appear to be shared by bromelain and phospholipase A2, thus suggesting that the IgE of patients reacting with such epitopes probably react with beta 1 --> 2 xylose, alpha 1 --> 3 fucose and/or alpha 1 --> 6 fucose.

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[110] - Asturias JA, Diéguez M, Ibarrola I, García R, De la Hoz B, Martínez A. Purified natural and recombinant Cup a 1 for diagnosis of Cupressus arizonica sensitization. Allergy Clin Immunol Int 2005;17(Suppl. 1):204

Background: C. arizonica is used in many Mediterranean countries for ornamental purposes in gardens and parks and also as wind and noise barriers. The low protein and high carbohydrate content found in Cupressaceae pollen extracts have hindered the availability of standardized extracts for diagnosis and immunotherapy. Allergenic extract of C. arizonica pollen contains a glycoprotein of 43 kDa (Cup a 1) which have an incidence of 82% among Arizona cypress-allergic patients. OBJECTIVE: To compare C. arizonica pollen extract and purified natural and recombinant Cup a 1 for their diagnostic value and to determine the importance of carbohydrate epitopes on diagnosis. METHODS: Cup a 1 was purified from C. arizonica pollen extract by hydrophobic interaction followed by gel permeation chromatography. Cup 1 encoding cDNA was cloned by RT-PCR, sequenced and subcloned in pTrc-HisB vector. rCup a 1 was produced in Escherichia coli and purified by metal-chelating chromatography. 27 patients with C. arizonica pollen allergy and 20 control subjects, 10 no-atopics and 10 with other no-related allergies, were included in the study. Skin prick test (SPT) was performed with C. arizonica pollen extract and natural and recombinant Cup a 1 at different concentrations (0.2, 2, 20, and 200 µg/ml). RESULTS: Both natural and recombinant Cup a 1 were able to elicit positive responses in vivo and in vitro. Skin response with purified allergens (both natural and recombinant) was dependent of allergen concentration. In SPT, sensitivity and specificity of nCup a 1 at 20 µg/ml were 81.5 and 94.4%, respectively and 74.1 and 94.4% for rCup a 1. At 200 µg/ml for both Cup a 1, the sensitivity was near 100% and the specificity was 83.3%. rCup a 1 exhibited in SPT a good correlation with nCup a 1 (r = 0.53; p<0.001). The sera of 24 and 23 out of 27 allergic patients contained IgE antibodies raised against nCup a 1 or total pollen extract respectively, while rCup a 1 reacted with only 11 patient sera. These different IgE binding values between nCup a 1 and rCup a 1 persisted after deglycosylation of natural allergen by treatment with peryodate. CONCLUSION: Either purified natural and recombinant Cup a 1 are sufficient for a reliable in vivo diagnosis of C. arizonica pollen allergy in most patients and induce comparable skin prick reactivity as the whole extract.

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[111] - Iacovacci P, Afferni C, Butteroni C, Pironi L, Puggioni EMR, Orlandi A, et al. Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils. Clin Exp Allergy 2002;32:1620-1627

Background Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. Objective : To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. Method s : Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. Result s : Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. Conclusion : A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy

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[112] - Palazzo P, Scala E, Bernardi ML, Mari A. Immunochemical and diagnostic impact of CCD-IgE reactivity on microarrayed allergens. Allergy 2007;62(suppl. 83):152-153

Background: The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the structural basis of what is known as IgE cross-reactive carbohydrate determinants (CCD). About 20% or more of allergic patients have been reported to produce specific anti-glycan IgE. CCD do not appear to cause clinical symptoms. We sought to establish the interference of anti-CCD IgE on a proteomic microarray system having both native and recombinant spotted allergens, including several glycoproteins. Methods: A population of 6155 subjects suspected for having an IgE-mediated disease has been screened using the multiplexed ISAC system (ISAC CRD 79b, VBC-Genomics, Austria). A serum was considered positive for CCD reactivity when a positive IgE reaction to Ana c 2 (bromelain) was detected. Twelve CCD-IgE positive subjects, having high IgE titers against Ana c 2, were tested on two more singleplex systems: CAP (Phadia, Sweden) and Immulite (Siemens, CA). IgE reactivity was detected for known glycoproteins rarely acting as true allergens, Ana c 2 and horseradish peroxidase, and to a purified glycopeptide from bromelain (MUXF3). A CCD-IgE adsorption was performed by using immobilized MUXF3 on CAP, then tested on ISAC system. Results: 4.2 % of the microarray tested population resulted positive for Ana c 2. A cluster reactivity was recorded for known glycoproteins, Ana c 2, Api m 1, but also for allergens never described for their glycan side chain IgE reactivity: Hor v 17, and Pla a 2. Allergens with a possible IgE reactivity for either CCD or protein epitopes (Lol p 1, Ole e 1, Cup a 1, Art v 1) were differently involved in this cluster reactivity. All the 12 tested subjects were recorded positive for tested glycoproteins and glycans on both in vitro systems. IgE adsorption of the tested serum exerted a complete inhibition of IgE reactivity to Ana c 2, Hor v 17, Api m 1, Pla a 2, Cup a 1, and Ole e 1. The lack of inhibition of other glycoallergens was scored as a prevalent IgE protein epitope reactivity. Conclusions: CCD-IgE are detected on microarrayed glycosilated allergens. MUXF3 from bromelain represent a suitable marker for CCD-IgE reactivity, and could be spotted on microarray system to detect specific CCD-IgE. To overcome the problem of CCD-IgE reactivity non-glycosylated recombinant glycoallergens could be used. At the moment we suggest to use a two step approach by adsorption or inhibition with MUXF3 glycopeptide or a highly purified glycoprotein.

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[113] - Malandain H, Fauquert JL, Tridon A, Susini de Luca H, Ughetto S, Cano Y. Peanut Allergy Diagnosis : Comparison of three Commercial Specific IgE Assays. J Allergy Clin Immunol 2006;117(2 suppl.):S33

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[114] - Petersen A, Johansen N. The outcome of determination of specific IgE is dependent upon the population being investigated. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1171

Background: Method comparisons are often used to demonstrate the characteristics of a new assay compared to an existing one. The aim of this study was to investigate what effect the choice of population and the choice of reference method may have for such comparisons, through measuring specific IgE (sIgE) in two populations in two different sIgE methods: the ADVIA Centaur (AC) and Pharmacia CAP (CAP) systems. Method: Serum samples from a Scandinavian population were tested for specific IgE against Japanese cedar (t17) and wall pellitory (w21) in the AC and CAP sIgE assays. In addition, t17 was tested on a sensitized Japanese population, and w21 was tested on a sensitized Spanish population for method comparison. Sensitivity and specificity was calculated for both methods, using the other as reference. Results: For the Scandinavian population, which may likely be assumed not to be truly sensitized to neither t17 nor w21, the concordance between the two methods was 92% for t17 and 73% for w21. For t17, AC showed a sensitivity of 87% and a specificity of 100% vs. CAP. Conversely for t17, CAP showed a sensitivity of 100% and a specificity of 83% vs. AC. For w21, AC showed a sensitivity of 49% and a specificity of 100% vs. CAP. Conversely for w21, CAP showed a sensitivity of 100% and a specificity of 63% vs. AC. All of the discordant Scandinavian samples were due to a positive response in the CAP system. These samples were tested for other sIgE reactivities. All AC negative and CAP positive samples were found to have sIgE response against one or more other allergens, primarily inhalants being present in the local environment. All of the discordant samples in t17 were tested positive on grass, tree and weed pollens prevalent in the Nordic countries. All of the discordant samples in w21 were tested positive on grass and tree pollens prevalent in the Nordic countries and/or Mugwort (w6) or other weed pollens. When tested on a more relevant population, the concordance between the two methods was 100% for t17 and 98% for w21. For t17, both AC and CAP showed a sensitivity and specificity of 100%. For w21, AC showed a sensitivity of 98% and specificity of 100% vs. CAP, whereas CAP showed a sensitivity of 100% and specificity of 96%. Conclusion: The selection and inclusion of patient samples for direct method comparison should be planned carefully with respect to bias towards one method and relevant patient exposure to allergens compared.

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[115] - Taniai M, Kayano T, Takakura R, Yamamoto S, Usui M, Ando S, et al. Epitopes on Cry j I and Cry j II for the human IgE antibodies cross-reactive between Cupressus sempervirens and Cryptomeria japonica pollen. Mol Immunol 1993;30:183-189

Forty sera from French patients allergic to Cupressus sempervirens pollen were tested for cross-reactivities against Cry j I, Cry j II (major allergens of Cryptomeria japonica pollen) and other pollen allergens from botanically related plants. Seventy-three per cent of the sera reacted with either Cry j I or Cry j II, or with both of them. These IgE cross-reactions were blocked effectively by mAb 046 (anti-Cry j I) or N26, T27 (anti-Cry j II), and weakly by mAbs 052, 027 and 026 (anti-Cry j I) Furthermore, the IgE antibodies in two sera, :40 and :11, bound to peptide fractions obtained from enzyme-digested Cry j I, and mAb 027 could also bind to the fractions. Analyses of the amino acid sequences of the peptides revealed that reactive peptides contained "NGNATPQLTKNAGVLTCSLSKR" sequence and the third residue N3 was glycosylated, however, when the N3 was not glycosylated, the IgE antibodies did not react, but mAb 027 could. The glycosylation of the N3 might be required for IgE-binding to the peptides. Sugar component on the N3 residue was found to be 0.4 mol galactose, 1.3 mol mannose, 0.8 mol fucose and 2.0 mol N-acetyl-glucosamine. Cross-reactivities against other pollen allergens from botanically related plants were found in most of the sera. However, many of these reactivities were detected by sandwich ELISA but not by an ELISA using allergen-coated plates, indicating that it is important to select an appropriate ELISA procedure in order to detect an allergen or an IgE antibody to an allergen.

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[116] - Ogawa H, Hijikata A, Amano M, Kojima K, Fukushima H, Ishizuka I, et al. Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergen Cry j I: relationship between the structures and antigenic epitopes of plant N-linked complex-type glycans. Glycoconj J 1996;13:555-566

The oligosaccharide structures of Cry j I, a major allergenic glycoprotein of Cryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz 1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch. Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides as Cry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing alpha 1-6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin and Clerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides on Cry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.

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[117] - di Felice G, Barletta B, Tinghino R, Pini C. Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens. Int Arch Allergy Immunol 2001;125:280-289

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity

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[118] - Kimura Y, Kuroki M, Maeda M, Okano M, Yokoyama M, Kino K. Glycoform Analysis of Japanese Cypress Pollen Allergen, Cha o 1: A Comparison of the Glycoforms of Cedar and Cypress Pollen Allergens. Biosci Biotechnol Biochem 2008;72:485-491

A Japanese cypress (Chamaecyparis obtusa) pollen allergen, Cha o 1, is one of the major allergens that cause allergic pollinosis in Japan. Although it has been found that Cha o 1 is glycosylated and that the amino acid sequence is highly homologous with that of Japanese cedar pollen allergen, the structure of N-glycans linked to Cha o 1 remains to be determined. In this study, therefore, we analyzed the structures of the N-glycans of Cha o1. The N-glycans were liberated by hydrazinolysis from purified Cha o 1, and the resulting sugar chains were N-acetylated and pyridylaminated. The structures of pyridylaminated N-glycans were analyzed by a combination of exoglycosidase digestion, two dimensional (2D-) sugar chain mapping, and electrospray ionization mass spectrometry analysis. Structural analysis indicated that the major N-glycan structure of Cha o1 is GlcNAc2Man3Xyl1Fuc1GlcNAc2 (89%), and that high-mannose type structures (Man9GlcNAc2, Man7GlcNAc2) occur as minor components (11%).

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[119] - Alisi C, Afferni C, Iacovacci P, Barletta B, Tinghino R, Butteroni C, et al. Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen. Allergy 2001;56:978-984

Background: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. Methods: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. Results: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF3 (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF3/GnGXF3 (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF3/Gn(GF)XF3 (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.

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[120] - Kimura Y, Kamamoto M, Maeda M, Okano M, Yokoyama M, Kino K. Occurrence of Lewis a epitope in N-glycans of a glycoallergen, Jun a 1, from mountain cedar (Juniperus ashei) pollen. Biosci Biotechnol Biochem 2005;69:137-144

We have determined the structures of N-glycans linked to major allergens in the mountain cedar (Juniperus ashei) pollen, Jun a 1. First, two kinds of the pollen glycoallergen (Jun a 1-A and Jun a 1-B) were purified from partially purified Jun a 1 by cation exchange chromatography. The N-glycans were liberated by hydrazinolysis from the two glycoallergens and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC from Jun a 1-A and Jun a 1-B respectively. The structures were determined by a combination of exo- and endo-glycosidase digestions, two dimensional sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS) analysis. Structural analysis indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-) occurs in the N-glycans of the pollen allergens.

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[121] - Maeda M, Kamamoto M, Hino K, Yamamoto S, Kimura M, Okano M, et al. Glycoform Analysis of Japanese Cedar Pollen Allergen, Cry j 1. Biosci Biotechnol Biochem 2005;69:1700-1705

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.

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[122] - Okano M, Kimura Y, Kino K, Michigami Y, Sakamoto S, Sugata Y, et al. Roles of major oligosaccharides on Cry j 1 in human immunoglobulin E and T cell responses. Clin Exp Allergy 2004;34:770-778

BACKGROUND: We have demonstrated that carbohydrates in Cry j 1, the major allergen of Cryptomeria japonica pollen, play a major role in promoting Cry j 1-specific Th2 response. However, little is known as to whether the carbohydrates directly participate in allergic responses . OBJECTIVE: We sought to determine whether Cry j 1-related oligosaccharides function as IgE and/or T cell epitopes. In addition, the regulatory effect of Cry j 1-related oligosaccharide on Cry j 1-specific T cell responses was investigated . METHODS: Two monovalent oligosaccharides largely found on Cry j 1, Manalpha1-6(Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc (M3FX), and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc (GN2M3FX) were prepared. Manalpha1-2Manalpha1-6(Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc (M9A) was used as control. Competitive inhibition ELISA for Cry j 1-specific IgE was performed using these oligosaccharides as inhibitors. In addition, T cell lines specific for Cry j 1 or purified protein derivative of Mycobacterium tubecurosis (PPD) were established, and cellular responses against these oligosaccharides were investigated in the presence or absence of the respective antigens . RESULTS: Overall, neither M3FX nor GN2M3FX displayed inhibitory effect on the binding between IgE and Cry j 1. In addition, M3FX did not by itself stimulate Cry j 1 or PPD-specific T cells. However, M3FX significantly inhibited Cry j 1-induced proliferation and IL-4 production in Cry j 1-specific T cells. Such an inhibitory effect was not seen in PPD-specific T cell responses . CONCLUSION: These results suggest that Cry j 1-related oligosaccharides are not major epitopes for IgE or T cells. However, these oligosaccharides have a novel potential to inhibit Cry j 1-specific T cell responses selectively.

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[123] - Altmann F. The role of protein-glycosylation in allergy. Int Arch Allergy Immunol 2007;142:99-115

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental 'immune therapy' ('glyco-specific immune therapy') exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single beta-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

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[124] - EXPO: Determinacion de la prevalencia de sensibilizacion a polenes mediante tecnicas de diagnostico molecular. ALK-Abello Ed., 2009, 86pp.

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[125] - Iacovacci P, Afferni C, Butteroni C, Pironi L, Puggioni EMR, Orlandi A, et al. Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils. Clin Exp Allergy 2002;32:1620-1627

Background Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. Objective : To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. Method s : Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. Result s : Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. Conclusion : A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy

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[126] - Okano M, Kino K, Takishita T, Hattori H, Ogawa T, Yoshino T, et al. Roles of carbohydrates on Cry j 1, the major allergen of Japanese cedar pollen, in specific T-cell responses. J Allergy Clin Immunol 2001;108:101-108

Background: Carbohydrates expressed on allergens are known to be important for allergenicity. However, little is known about whether the carbohydrates drive the TH2 response. Objective: We sought to determine a role for carbohydrates expressed on Cry j 1, which is the major allergen of Cryptomeria japonica pollen and causes the most prevalent pollinosis in Japan, in in vitro cellular responses. Methods: Carbohydrates on Cry j 1 were destroyed by periodate-oxidation under mild conditions. Proliferative responses and cytokine productions against native, periodate-treated, and mock-treated Cry j 1 were compared in peripheral blood mononuclear cells, Cry j 1ˆspecific T-cell lines, and clones from patients with Japanese cedar pollinosis. Results: We found that peripheral blood mononuclear cells from patients with Japanese cedar pollinosis displayed a significant decrease in proliferation and IL-5 production in response to periodate-treated Cry j 1 in comparison with native and mock-treated Cry j 1. Decreased proliferative responses against periodate-treated Cry j 1 were also seen in polyclonal T-cell lines, and the responses showed a heterogeneity. In addition, Cry j 1ˆspecific CD4+ T-cell clones also displayed a significant decrease in proliferation and IL-4 and IL-5 production˜but not IFN- production˜in comparison with the control antigens. However, most of the clones showed decreased but positive proliferative responses against periodate-treated Cry j 1. Blockade of the mannose receptor had no effect on cellular responses. Conclusion: The results suggest that carbohydrates on Cry j 1 play a major role in promoting Cry j 1ˆspecific TH2 response in vitro, though they are not major targets as T-cell epitopes.

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Chamaecyparis obtusa	Cyprès du Japon	Cha o 1	Cha o 2
Cryptomeria japonica	Cèdre du Japon	Cry j 1	Cry j 2	Cry j 3	Cry j 4
Cupressus arizonica	Cyprès d’Arizona	Cup a 1	Cup a 2	Cup a 3	Cup a 4
Cupressus sempervirens	Cyprès commun	Cup s 1		Cup s 3
Juniperus ashei	Sabine	Jun a 1	Jun a 2	Jun a 3
Juniperus communis	Genévrier commun	Jun c 1
Juniperus oxycedrus	Genévrier oxycèdre	Jun o 1			Jun o 4
Juniperus rigida	Genévrier japonais			Jun r 3
Juniperus virginiana	Red cedar	Jun v 1		Jun v 3	Jun v 4
Taxodium distichum	Cyprès chauve		Tax d 2
Thuja occidentalis	Thuya du Canada			Thu oc 3
Thuja plicata	Thuya géant	Thu p 1

Les allergènes des Cupressacées

Réactions croisées entre pollens de Cupressacées

Réactions croisées entre Cupressacées et d’autres pollens

Cupressacées et allergie alimentaire

Pollens de Cupressacées et latex

Diagnostic d’une sensibilisation aux Cupressacées

Pollens de Cupressacées et CCD

Rubriques

Dans la même rubrique

Le bouleau

Les Fagales

Les Oléacées

Les Pinacées

Le platane