Ole e 3, a Candidate for in vivo Diagnosis of Polcalcin Sensitization.

INTRODUCTION: Polcalcins belong to the family of calcium-binding proteins. They are ubiquitous in the plant kingdom and highly conserved, which leads to these panallergens showing a high degree of inter-cross-reactivity. They are responsible for allergic polysensitization, and therefore, their diagnosis is necessary for correct selection of immunotherapy. The objectives were to develop a method to purify native polcalcin with intact allergenic properties and to validate its use for diagnosis of polcalcin sensitization. METHODS: Ole e 3 was purified by immunoaffinity chromatography using anti-rChe a 3 polyclonal antibodies and identified by mass spectrometry. Calcium-binding assays were performed in immunoblot and ELISA assays. Diagnostic capacity of Ole e 3 was analyzed by ELISA and compared to ImmunoCAP with sera from a pollen-sensitized population. Cross-reactivity with other polcalcins was investigated by ImmunoCAP inhibition. RESULTS: Immunogenicity of purified Ole e 3 was not affected by the addition of calcium. However, the presence of a calcium chelator agent completely inhibited IgG binding by immunoblot and produced a 32.3% reduction in IgE binding by ELISA. Ole e 3 enabled diagnosis of polcalcin-sensitized patients, and a good correlation was revealed with ImmunoCAP. A 50% inhibition in IgE binding was obtained with 2.8 ng of Ole e 3 for rBet v 4 and 3.9 ng for rPhl p 7. DISCUSSION/CONCLUSION: Native Ole e 3 was purified by maintaining its allergenic properties. This innovative method enables obtaining this active native allergen to be used for in vivo diagnosis of polcalcin sensitization.

Prevalence of allergic sensitization to conifer pollen in a high cypress exposure area.

BACKGROUND: Sensitization to Pinales (Cupressaceae and Pinaceae) has increased dramatically in recent years. The prevalence of sensitization in different geographic areas is related to exposure to specific pollens. OBJECTIVES: To investigate the prevalence of allergy to different conifer pollens, describe the characteristics of patients with such allergy, and identify the involved allergens. METHODS: Patients were recruited at five hospitals near Madrid. Extracts from conifer pollen were prepared and used in skin-prick testing. Wheal sizes were recorded, and serum samples obtained from patients with positive reactions to Cupressus arizonica and/or Pinus pinea. The specific immunoglobulin E value to C. arizonica and Cup a 1 was determined. Individual immunoblots for each patient and with a pool of sera were performed. Allergenic proteins were sequenced by using liquid chromatography-tandem mass spectrometry. RESULTS: Of 499 individuals included in the study, 17 (14%) had positive skin-prick test results to some conifer pollen extracts. Sixty-four patients had positive results to C. arizonica (prevalence 12.8%) and 11 had positive results to P. pinea (2.2%). All the patients had respiratory symptoms (61.4% during the C. arizonica pollination period), and 62.9% had asthma. Approximately 86% of the patients had positive specific immunoglobulin E results to C. arizonica and 92.3% had positive results to Cup a 1. Fourteen different bands were recognized by immunoblot; the most frequent bands were those detected at 43, 18, 16, and 14 kDa. All sequenced proteins corresponded to Cup a 1. CONCLUSION: Allergy to conifer pollen could be considered a relevant cause of respiratory allergy in central Spain. Asthma was more frequent than in other studies. We only identified Cup a 1 as involved in sensitization.

Recently introduced foods as new allergenic sources: sensitisation to Goji berries (Lycium barbarum).

Goji berries (GB) have been introduced in Western diet. Preliminary reports have demonstrated its allergenic capacity. The objectives of the study were to investigate the frequency of sensitisation and the allergens involved. 566 individuals, with respiratory or cutaneous symptoms were skin-prick tested with GB extract. Thirty three were positive (5.8%). 94% were sensitised to other allergens. Specific IgE to GB, peach, tomato and nut-mix was measured. Thirteen individuals from 24 available sera (54.2%) had positive specific IgE. 92.3% of GB positive patients were positive to peach. Seven individuals recognised 8 bands and six recognised a 7kDa band. This band was identified as a LTP by MS/MS. Cross-reactivity was demonstrated with tomato, tobacco, nutmix, Artemisia pollen and purified Lyc e 3 and Pru p 3. GB are a new allergenic source with high prevalence of sensitisation. LTP seems to be the major allergen involved in sensitisation and cross-reactivity.

Assessing degree of flowering implicates multiple Chenopodiaceae/Amaranthaceae species in allergy.

BACKGROUND: IgE-mediated sensitization to the Chenopodiaceae/Amaranthaceae families is a cause of allergic symptoms in arid areas. Salsola kali and Chenopodium album are considered the main species responsible; however, there is a discrepancy between the pollination period of these two plants and clinical symptoms. The objectives of this study were to identify new Chenopodiaceae/Amaranthaceae members with sensitization capacity and to correlate symptoms, pollen counts and degree of flowering of different species. METHODS: A total of 37 individuals monosensitized to S. kali and C. album were included in the study. All patients recorded daily symptom scores between May and October 2007. Extracts from Chenopodium (album, vulvaria and murale), Salsola (kali, vermiculata, and oppositifolia), Bassia scoparia, Atriplex (patula and halimus) and Amaranthus (deflexus and muricatus) were manufactured and used in skin prick tests (SPTs). Protein content and IgE binding were assessed for each extract. Pollen counts and degree of flowering (based on the Orshan specific semiquantitative method) were assessed weekly. RESULTS: Symptom scores demonstrated a positive correlation with pollen counts even outside the pollination period of S. kali. Positive SPTs were obtained with all 11 species tested, which showed common proteins with IgE-binding capacity. Different species flowered at different times during the pollen season. CONCLUSION: Different taxonomically related species of Chenopodiaceae/Amaranthaceae can induce allergic sensitization and should be considered for use in diagnosis and treatment. Degree of flowering is a complementary method for assessing pollination that could be used for botanical families with indistinguishable pollen grains.

Protein cryoprotective activity of a cytosolic small heat shock protein that accumulates constitutively in chestnut stems and is up-regulated by low and high temperatures.

Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.