Characterization of a 7 kDa pollen allergen belonging to the gibberellin-regulated protein family from three Cupressaceae species.

BACKGROUND: Severe allergy to fruits mediated by a 7 kDa allergen belonging to the gibberellin-regulated protein (GRP) family is known to be associated with Cupressaceae pollinosis. OBJECTIVE: To identify and characterize Cupressaceae pollen allergens involved in GRP-related fruit allergy. METHODS: Pru p 7-related proteins from pollen of Cupressus sempervirens, Juniperus ashei and Cryptomeria japonica were identified using a rabbit anti-Pru p 7 antiserum, purified chromatographically and sequenced by mass spectrometry and bioinformatic comparisons. The C sempervirens protein was produced as a recombinant allergen in Pichia pastoris. IgE antibody binding to pollen GRP proteins was analysed in a peach allergic (n = 54) and a cypress pollen allergic (n = 88) patient population from southern France using ImmunoCAP. RESULTS: In each of the three Cupressaceae species studied, a 7 kDa pollen protein related to Pru p 7 was identified and found to comprise an amino acid sequence of 63 residues in length, 92%-98% identical to each other and 67%-68% identical to Pru p 7. The C sempervirens, J ashei and C japonica GRP allergens have been officially recognized by the WHO/IUIS Allergen Nomenclature Sub-Committee and named Cup s 7, Jun a 7 and Cry j 7, respectively. Recombinant Cup s 7 showed IgE antibody binding capacity comparable to that of the purified natural allergen. Among 51 peach allergic subjects sensitized to Pru p 7, substantially higher levels of IgE to Cup s 7 than to Pru p 7 were found. Further, the pollen protein was able to completely outcompete IgE binding to Pru p 7, while the reverse competition effect was modest, consistent with primary sensitization by the pollen allergen. CONCLUSION AND CLINICAL RELEVANCE: Pru p 7-related pollen allergens from three Cupressaceae species have been characterized and may become useful for the identification of pollinosis patients at risk of developing severe fruit allergy.

Hinokitiol reduces matrix metalloproteinase expression by inhibiting Wnt/ß-Catenin signaling in vitro and in vivo.

OBJECTIVE: In this study, we investigated the effects of hinokitiol on matrix metalloproteinase (MMP)-1, -3, -13, collagen type II (Col2a1) and ß-catenin expressions in rat chondrocytes induced by interleukin-1ß and in an experimental rat model induced by intra-articular injection of mono-iodoacetate (MIA) into the knee. METHODS: Chondrocytes were cultured from the articular cartilage of 2-week-old rats. Passaged chondrocytes were pretreated with hinokitiol for 2h followed by co-incubation with IL-1ß for 24h. Quantitative real-time polymerase chain reaction and Western blotting were used to assess the expression of MMP-1, -3, -13, Col2a1 and ß-catenin. Chondrocytes were also treated with Licl, Dickkopf-1, and/or hinokitiol for 24h, the MMP-1, -3, -13 and ß-catenin protein levels determined by Western blotting. The in vivo effects of hinokitiol were assessed by morphological and histological analyses following MIA injection. RESULTS: Hinokitiol inhibited IL-1ß-stimulated MMP-1,-3 and -13 expressions and IL-1ß-induced activation of intracellular ß-catenin proteins in cultured chondrocytes. In vivo, morphological and histological examinations demonstrated that hinokitiol significantly ameliorated cartilage degeneration. CONCLUSIONS: Hinokitiol is an effective anti-inflammatory reagent that acts by inhibiting the Wnt/ß-catenin signaling pathway and could be a promising therapeutic agent for the prevention and treatment of osteoarthritis.

Short-term effects of airborne pollens on asthma attacks as seen by general practitioners in the Greater Paris area, 2003-2007.

AIMS: To investigate for the first time the short-term effects of airborne pollen counts on general practitioner (GP) consultations for asthma attacks in the Greater Paris area between 2003-2007. METHODS: Counts were available for common pollens (Betula, Cupressa, Fraxinus and Poaceae). Weekly data on GP visits for asthma attacks were obtained from the French GP Sentinel Network. A quasi-Poisson regression with generalised additive models was implemented. Short-term effects of pollen counts were assessed using single and multi-pollen models after adjustment for air pollution and influenza. RESULTS: A mean weekly incidence rate of 25.4 cases of asthma attacks per 100,000 inhabitants was estimated during the study period. The strongest significant association between asthma attacks and pollen counts was registered for grass (Poaceae) in the same week of asthma attacks, with a slight reduction of the effect observed in the multi-pollen model. Adjusted relative risk for Poaceae was 1.54 (95% CI: 1.33-1.79) with an inter-quartile range increase of 17.6 grains/m3 during the pollen season. CONCLUSIONS: For the first time, a significant short-term association was observed between Poaceae pollen counts and consultations for asthma attacks as seen by GPs. These findings need to be confirmed by more consistent time-series and investigations on a daily basis.

Cupressaceae pollen grains modulate dendritic cell response and exhibit IgE-inducing adjuvant activity in vivo.

Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.

Associations among pollen sensitizations from different botanical species in patients living in the northern area of Madrid.

OBJECTIVE: To determinate the existence of associations among sensitizations to antigens produced by pollen grains of different botanical species as assessed by skin prick tests in patients with respiratory disorders. METHODS: Six hundred twenty nine consecutive patients living in the northern area of Madrid who underwent clinical evaluation because of rhinoconjunctivitis, and/or asthma were studied. All patients were tested with a skin prick test using a battery of inhalants including pollens, dust mites, molds and danders. The exploratory multivariate technique of Multiple Correspondence Analysis was used to compare the homogeneity of sensitizations between groups. Of the 629 patients, 459 (73.0%) had positive skin prick tests to pollen and were selected as the study group. RESULTS: The most prevalent pollen sensitization was to Gramineae pollen (83.7%) followed by Oleaceae sensitisation (75.8%). Multiple Correspondence Analysis revealed the existence of an association among pollen sensitizations, showing that they clustered two groups: sensitizations to Gramineae, Oleaceae, Cupressaceae, Chenopodiaceae, Plantaginaceae (group I), and sensitizations to Betulaceae, Platanaceae, Compositae (group II). Sensitization to Parietaria was not included in any of the sensitization groups and showed an independent behaviour. CONCLUSION: Pollen sensitizations in our area cluster into two association groups which have not previously been reported.

Effect of air temperature on forecasting the start of Cupressaceae pollen type in Ponferrada (Leon, Spain).

In order to survive periods of adverse cold climatic conditions, plant requirements are satisfied by means of physiological adaptations to prevent cells from freezing. Thus, the growth of woody plants in temperate regions slows down and they enter into a physiological state called dormancy. In order to identify the chilling and heat requirements to overcome the dormancy period of Cupressaceae pollen type in the south of Europe, we have carried out our study with aerobiological data from a 10-year (1996-2005) period in Ponferrada, León (Spain). For the chilling requirements the best result was with a threshold temperature of 7.1 degrees C and an average of 927 CH. Calculation of heat requirements was carried out with maximum temperature, with 490 growth degree days (GDD) needed, with a threshold temperature of 0 degrees C. We have used the 2002-2003, 2003-2004 and 2004-2005 periods in order to determine the real validity of the model. We have not used these years in developing the models. The dates predicted differ in only a few days from those observed: in 2002-2003 there was a difference of 11 days, in 2003-2004 predicted and observed dates were the same, but in 2004-2005 the difference obtained was of 43 days.

Biochemical safety evaluation of transgenic rice seeds expressing T cell epitopes of Japanese cedar pollen allergens.

UNLABELLED: Transgenic rice seeds, which express a hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, have been shown to function as an effective edible vaccine for the control of pollen allergen-induced responses. In this study, we characterized biochemical properties of transgenic seeds expressing the 7Crp peptide. The levels of chemical compositions, such as carbohydrate, protein, lipid, amino acid, fatty acid, mineral, and vitamin, were substantially equivalent between transgenic 7Crp and its nontransgenic counterpart seeds. The contents of three major allergenic proteins in transgenic seeds were not enhanced by expression of the 7Crp peptide when compared with those of nontransgenic seeds. The 7Crp peptide expressed in seeds was susceptible to simulated gastric/intestinal fluids. N-Glycosylation was not observed in the 7Crp peptide sequence. These results indicate that transgenic 7Crp seeds are substantially equivalent to nontransgenic parental seeds except for the presence of the 7Crp peptide. KEYWORDS: Food safety assessment; transgenic rice seed; edible vaccine; peptide-based immunotherapy; Japanese cedar pollinosis.

Aerobiological and allergenic analysis of cupressaceae pollen in Granada (Southern Spain).

Cupressaceae pollen has been cited in recent years as one of the major airborne allergens of the Mediterranean region, prompting us to conduct an exhaustive analysis on the aerobiological behaviour of this pollen in the Iberian Peninsula and the repercussion that it has had on the atopic population. The aerobiological study, performed from 1996 to 2003 in the city of Granada (S. Spain), used a volumetric Hirst collector. The results indicate that this pollen is present in the air most of the year, registering a high incidence during the winter months. This type of pollen behaved irregularly in the air, fluctuating yearly, seasonally, and within the same day. Temperature and humidity were the parameters that most directly influence the variability of this allergen, while rainfall prior to flowering increased pollen production. The predictive models used estimated a high percentage of the levels reached over the short term by this pollen in the atmosphere of Granada. The clinical study performed with atopic patients showed that some 30% of the population with pollinosis are sensitive to Cupressaceae pollen, affecting people of both genders equally. On the other hand, the most sensitive age group was 21-40 years of age, while children and the elderly registered almost negligible values. Most of the sensitive subjects resided within the city or in the metropolitan area, where environmental pollution reached high levels, while the pathology was found to be less frequent in rural zones. The most frequent symptoms were upper-respiratory ailments and an asthmatic profile.

Efficacy of sublingual coseasonal immunotherapy with a monomeric allergoid in Cupressaceae pollen allergy--preliminary data.

BACKGROUND: Respiratory allergy to conifers pollens is a growing phenomenon all over the world (Mediterranean area, USA, Japan). In France and Italy Cupressus sempervirens is the commonest offender. OBJECTIVE: To check the efficacy of specific immunotherapy by the sublingual route using a monomeric allergoid of Cupressus Arizonica pollen. MATERIAL AND METHODS: 20 patients and 10 controls were randomly selected. The diagnosis was assessed on a clinical hivernal symptomatology of allergic rhinitis, and/or asthma, positive skin test and RAST, and nasal provocation test to Cupressus arizonica. The extract was a monomeric allergoid titrated in allergenic units (AU). The treatment was coseasonal from the beginning of december to the end of march. It was not conducted double blind. RESULTS: The evaluation of the allergenic potency of native and modified Cupressus arizonica extracts have shown that the modified extract has a decreased skin test IgE binding capacity. According to symptom, drug consumption scores and nasal provocation test, there was a statistical improvement in the active group (compared to the control group) without any side effect. DISCUSSION: The use of a modified Cupressus arizonica extract, which is easier to prepare than Cupressus sempervirens and which is a monomeric allergoid, has shown that by the sublingual route, this therapeutic approach is safe and that the first results were satisfactory without any side effect. This good tolerance was anticipated by the fact that, checked on 16 volunteers by skin test, the allergoid gave a less important local reaction on skin testing than the native extract. CONCLUSION: This study has shown that: The KCNO modified Cupressus arizonica extract has a reduced IgE binding capacity; This monomeric allergoid is safe and effective administrated by the oromucosal route in clinical respiratory allergy to Cupressaceae and devoid of any side effect.

Localisation of a carbohydrate epitope recognised by human IgE in pollen of Cupressaceae.

The objectives of the present study were: (1) to localise, at the subcellular level, the allergens in pollen of Cupressaceae species, using a monoclonal antibody (mAb 5E6) that is specific for carbohydrate epitopes of allergenic components of Cupressus arizonica pollen extract; (2) to determine whether the glycidic epitope recognised by mAb 5E6 was present in pollen of allergenic species taxonomically unrelated to Cupressaceae; and (3) to determine whether human IgE purified from monosensitive patients recognises the same epitope as mAb 5E6 in Cupressaceae pollen. Immunogold labelling of mAb 5E6 showed a high density of gold particles on the orbicules, supporting the hypothesis that they are important vectors of allergens. A high density was also found on the exine and in the cytoplasm, with the latter finding confirming that fragments of pollen ruptured under humid conditions can represent a vector. The glycidic epitope recognised by mAb 5E6 was detected in all of the species taxonomically unrelated to Cupressaceae, although with varying density. Human IgE recognised the same epitope as mAb 5E6. These findings are consistent with observations of diffuse allergenic cross-reactivity among various allergens. The in situ localisation of a common epitope recognised by both a monoclonal antibody and human IgE could be of importance in immunotherapy.

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

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

An epidemiological survey of Cupressaceae pollenosis in Italy.

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.

Efficacy of sublingual specific immunotherapy in Cupressaceae allergy using an extract of Cupressus arizonica. A double blind study.

BACKGROUND: non-injective routes of immunotherapy in respiratory allergy have being proposed as an alternative to conventional immunotherapy. We carried out a study to evaluate the clinical efficacy and effects sublingual immunotherapy (SLIT) in patient with Cupressaceae pollen respiratory allergy. METHODS: twenty patients with Cupressaceae pollen (C. sempervirens) rhinoconjunctivitis, mild asthma or both were randomly chosen for sublingual immunotherapy (10 patients) or placebo treatment (10 patients) using a double blind placebo controlled technique. We have used an extract of Cupressus arizonica because of its better stability in solution and its crossreactivity with Cupressus sempervirens. The patients underwent treatment for 12 months (from april 1999 to april 2000). Symptoms and drug scores as well as nasal provocation tests were recorded. The pollen counts were carried out, during all the same period. RESULTS: we found significantly lower symptom scores (p < 0.05) and drug consumption scores (p < 0.05) in the immunotherapy group than in the placebo group, during the pollen season, after the first year of therapy. Besides threshold of allergen reactivity in nasal provocation tests was increased in the active group (p < 0.01). No untoward reactions have been observed. CONCLUSIONS: SLIT with Cupressus arizonica (pollen extracts) produced, after one year treatment, a significant improvement of allergic symptoms and a decrease of drug consumption scores and allergen-specific nasal reactivity. SLIT with Cupressus arizonica appeared to be effective and safe in the treatment of Cupressaceae pollen respiratory allergy.