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Phl P (phl + p)
Selected AbstractsAllergen content of grass pollen preparations for skin prick testing and sublingual immunotherapyALLERGY, Issue 10 2009I. Sander Background:, The allergen content of diagnostics and immunotherapeutics is crucial for effective diagnosis and treatment. The aim of this study was to quantify and compare the allergen content of different grass pollen preparations for skin prick testing and sublingual immunotherapy (SLIT). Methods:, Five skin prick test (SPT) solutions and 10 sublingual immunotherapeutics were analysed for protein and allergen concentration by Bradford assay, inhibition of IgE-binding to Phleum pratense ImmunoCAPs and content of the main allergen Phl p 5 by two-site enzyme immunoassay. In addition, the grass pollen preparations were compared by sodium dodecyl sulfate,polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses. Results:, Protein concentrations of SPT solutions ranged from 15 to 427 ,g/ml, and Phl p 5 concentrations ranged from 0.15 to 18.3 ,g/ml. The ranking of SPT solutions concerning Phl p 5 content and IgE inhibition capacity was the same, and the ranking of protein and allergen content was closely correlated (r = 0.9). Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 ,g, Phl p 5 content ranged from 0.2 to 21.6 ,g. IgE inhibition capacity of the maintenance doses was closely correlated to their Phl p 5 and protein content. SDS-PAGE and immunoblots confirmed the differences in protein and allergen content. Conclusions:, Grass pollen preparations for SPT and SLIT varied greatly concerning protein and allergen content. Whereas this result corresponds to previous analyses results of SPT solutions, it was the first comparison of grass pollen immunotherapeutics. For diagnosis and therapy, these differences should be taken into account. [source] Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccinationALLERGY, Issue 4 2009T. Ball Background:, The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy. Methods:, A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice. Results:, Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils. Conclusion:, We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination. [source] Heterogeneity of commercial timothy grass pollen extractsCLINICAL & EXPERIMENTAL ALLERGY, Issue 8 2008M. Focke Summary Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources. Objective To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity. Methods Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients. Results The allergen extracts showed broad variations in protein compositions and amounts (24.1,197.7 ,g/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32,384 ng/mL; Phl p 2: 1128,6530 ng/mL, Phl p 5: 40,793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients. Conclusions Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens. [source] Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5CLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2006Å. Marknell DeWitt Summary Background Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein. Objective To clone and characterize timothy grass pollen allergen Phl p 4. Methods Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3,-and 5,-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies. Results Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner. Conclusions Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4. [source] Quantification of group 5 grass pollen allergens in house dustCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000B. Fahlbusch Background It is widely known and accepted that grass pollen is a major outdoor cause of hay fever. However, it is of virtual importance for grass pollen allergic patients with symptoms all the year round to know the concentration of grass pollen allergens in their homes. Objective The main objective of this study was to quantify the amount of grass pollen allergen in mass units (,g Phl p 5) in dust settled indoors and to detect the distribution of allergenic activity in different sampling locations of homes. Furthermore, we studied the seasonal fluctuation of allergen content in dust samples. Methods We adapted the two site binding assay for detection of group 5 grass pollen allergens in samples from randomly selected homes in Hamburg (n = 371), Erfurt (n = 396), Hettstedt (n = 353), Zerbst (n = 289) and Bitterfeld (n = 226), Germany. Dust samples were collected from floor of living room (LR), bedroom (BR) or children's room (CR) and mattress (MA) during period of June 1995 to August 1998. The amount of the major grass group 5 allergens was detected in ,g/g dust. Results Phl p 5 was detected in 67% of the samples analysed (n = 4760). The range was between undetectable (< 0.03 ,g/g dust) and 81 ,g/g dust. Phl p 5 levels were significantly higher in the dust from LR (geometric mean 0.117 ,g/g dust) or BR/CR floors (geometric mean 0.098 ,g/g dust) than in mattresses (geometric mean 0.043 ,g/g dust). We observed seasonal fluctuation of indoor Phl p 5 levels with peak in June but also annual differences. Thus Phl p 5 content indoors reflects also the different quantities of pollen counts of annual courses. During pollination period we found two times higher Phl p 5 levels (0.172 ,g/g dust, P < 0.001) than outside of grass pollination season (0.095 ,g/g dust). The indoor Phl p 5 levels outside of season seem to be independent of pollination before. We suppose that settled pollen grains or allergenic material from outdoor particles carried indoors via footwear and clothes accumulates in house dust. Conclusion Although we not known how the allergens in settled dust are equilibrated with those in the air, the considerable high level of Phl p 5 in indoor dust even during periods when no grass pollen is present in the atmosphere may be an important cause of pollen-allergy symptoms outside of season. [source] | ||||||||||