The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Rudolf Valenta - One of the best experts on this subject based on the ideXlab platform.
-
detection of genuine grass pollen sensitization in children by skin testing with a recombinant grass pollen hybrid
Applied Immunohistochemistry & Molecular Morphology, 2019Co-Authors: Nikolaos Douladiris, Rudolf Valenta, Victoria Garib, Margit Focketejkl, Nikolaos G Papadopoulos, Birgit LinhartAbstract:Background Skin testing represents a commonly used first diagnostic method in clinical practice, but allergen extracts may vary in composition and often contain cross-reactive allergens and therefore do not always allow the precise identification of the sensitizing allergen source. Our aim was to investigate the suitability of a single recombinant hybrid molecule, consisting of the four major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Phl p 6) for in vivo diagnosis of genuine grass pollen allergy in children suffering from pollinosis. Methods Sixty-four children aged from 6 to 17 years with a positive skin reaction and/or specific IgE to grass pollen extract and respiratory symptoms of pollinosis as well as 9 control children with allergy to other allergen sources were studied. SPT was performed with the recombinant hybrid, the four recombinant timothy grass pollen allergens, and grass pollen extract. Specific IgE reactivity to 176 micro-arrayed allergen molecules was determined using ImmunoCAP ISAC technology. IgE reactivity to the hybrid was detected by non-denaturing RAST-based dot blot assay. Results Genuine grass pollen sensitization was confirmed in 94% of the children with positive SPT to grass pollen extract by SPT and IgE reactivity to the hybrid. The four hybrid-negative children showed IgE reactivity to cross-reactive allergens such as Phl p 4, Phl p 11, and Phl p 12 and had also sensitizations to pollen allergens from unrelated plants. Conclusions The recombinant hybrid molecule represents a useful tool for in vivo diagnosis of genuine grass pollen sensitization.
-
heterogeneity of commercial timothy grass pollen extracts
Clinical & Experimental Allergy, 2008Co-Authors: Max Focke, Katharina Marth, Sabine Flicker, Rudolf ValentaAbstract: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 (24.1‐197.7mg/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: 32‐384ng/mL; Phl p 2: 1128‐6530ng/mL, Phl p 5: 40‐793ng/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.
-
Skin test diagnosis of grass pollen allergy with a recombinant hybrid molecule
The Journal of Allergy and Clinical Immunology, 2007Co-Authors: C. Metz-favre, Margarete Focke-tejkl, Ashok Purohit, Birgit Linhart, Frederic De Blay, Rudolf Valenta, Gabrielle PauliAbstract:Background A recombinant hybrid molecule (HM) consisting of 4 major allergens from timothy grass (Phl p 1, 2, 5, and 6) was expressed in Escherichia coli , purified, and characterized regarding its immunologic properties. Objective We sought to determine whether the recombinant HM can be used for the diagnosis of grass pollen allergy by means of skin testing. Methods Skin prick testing was performed in 32 patients with grass pollen allergy and in 9 control individuals by using increasing concentrations (4, 12, 36, and 108 μg/mL) of the HM and using commercial grass pollen extract. Specific IgE reactivities against the HM, grass pollen extract, and a panel of purified grass pollen allergens (recombinant Phl p 1, 2, 5, 6, 7, 12, and 13 and natural Phl p 4) were measured by means of ELISA, and timothy grass pollen–specific IgE levels were determined by using ImmunoCAP. Results Grass pollen allergy was diagnosed in all patients by means of skin testing with the HM. No false-positive skin test responses were obtained in the control individuals. There was an excellent correlation between IgE levels obtained with the HM and natural grass pollen extract measured by means of ELISA ( r = 0.98, P r = 0.98, P Conclusions The recombinant HM permitted accurate and specific in vivo diagnosis of grass pollen allergy in all tested patients. It can be considered a well-defined tool for the diagnosis and perhaps for immunotherapy of grass pollen allergy. Clinical implications A recombinant HM can replace traditional allergen extracts for skin test–based diagnosis of grass pollen allergy.
-
group 13 allergens as environmental and immunological markers for grass pollen allergy studies by immunogold field emission scanning and transmission electron microscopy
International Archives of Allergy and Immunology, 2005Co-Authors: Monika Grote, Ines Swoboda, Rudolf Valenta, Rudolf ReicheltAbstract:BACKGROUND: Polygalacturonases were recently identified as important grass pollen allergens and designated group 13 allergens. The objective of the present study was to investigate the presence of group 13 grass pollen allergens in different grass species, their release and ultrastructural location in dry and hydrated grass pollen. METHODS: Nitrocellulose-blotted allergen extracts from 12 wild and cultivated grass genera were probed with a rabbit antiserum raised against purified recombinant timothy grass pollen allergen, Phl p 13. The release kinetics of Phl p 13 from timothy grass pollen hydrated for 0.5 min to 3 h were analyzed by immunoblotting. Phl p 13 was localized in dry and hydrated grass pollen grains by immunogold field emission scanning and transmission electron microscopy. RESULTS: Group 13 allergens were detected in all 12 wild and cultivated grass genera representing the major subfamilies of the Poaceae. Ultrastructurally, the allergen was located in the wall and in the cytoplasm of timothy grass pollen grains. In the cytoplasm, Phl p 13 was associated with polysaccharide particles and as yet undescribed stacks of microtubule-like structures. After hydration in rain water, pollen grains expel cytoplasmic particles of respirable size containing Phl p 13, which becomes detectable in aqueous supernatants already after 0.5 min. CONCLUSIONS: Group 13 allergens represent one set of marker allergens which specifically occur in pollen of the major grass subfamilies and are rapidly released in association with respirable particles after pollen hydration. They may be considered as environmental markers for grass pollen exposure and group 13-specific IgE antibodies as immunological markers for genuine grass pollen sensitization.
-
Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 2002Co-Authors: Å. Marknell Dewitt, Susanne Spitzauer, Rudolf Valenta, Wolfgang R. Sperr, Peter Valent, Verena Niederberger, P Lehtonen, Jonas LidholmAbstract:Molecular and immunogical characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11
Otto Scheiner - One of the best experts on this subject based on the ideXlab platform.
-
T Cell Epitopes of Phl p 1, Major Pollen Allergen of Timothy Grass (Phleum pratense)
Advances in experimental medicine and biology, 1996Co-Authors: Siegfried Schenk, Markus Susani, Sylvia Laffer, Rudolf Valenta, Michael Duchêne, Otto Scheiner, Heimo Breiteneder, Nader Najafian, Gottfried Fischer, Dietrich KraftAbstract:Recently several group I grass pollen allergens, such as Phl p 1 from timothy grass (Phleum pratense), some isoforms of Loi p 1 from rye-grass (Lolium perenne), and Sec c 1 from rye (Secale cereale; S. Laffler, unpublished results) have been characterized by molecular cloning techniques1–3 and were found to display a high degree of amino acid (AA) sequence-similiarity. T lymphocytes play an important role in the induction of IgE-synthesis, and therefore in the development of Type I allergy4–6. In this study humoral and cellular responses to recombinant Phl p 1 (from timothy grass) were analyzed. T cell lines (TCL) and T cell clones (TCC) were established to determine T cell epitopes of this allergen. IgE-crossreactivity among pollen allergens of different grass species has been observed on the IgE-level1. We investigated T cell-crossreactivity among grass group I allergens. The knowledge of major and/or crossreacting T cell epitopes of grass pollen allergens2,7 is important for the development of vaccines for immunotherapy (IT) in the future.
-
cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli : comparison with birch pollen profilin
Biochemical and biophysical research communications, 1994Co-Authors: Rudolf Valenta, Tanja Ball, Susanne Vrtala, Michael Duchêne, D. Kraft, Otto ScheinerAbstract:Abstract Profilin, an actin-binding protein,was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass ( Phleum pratense ) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a β-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.
-
IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens
The Journal of allergy and clinical immunology, 1994Co-Authors: Sylvia Laffer, Susanne Vrtala, Michael Duchêne, Otto Scheiner, Ronald Van Ree, Dietrich Kraft, Rudolf ValentaAbstract:Abstract A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens. In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed. Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen–specific IgE was estimated. Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I . The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens. The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen–specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy. (J ALLERGY CLIN IMMUNOL 1994;94:88-94.)
-
Complementary DNA cloning of the major allergenPhl p I from timothy grass (Phleum pratense); recombinantPhl p I inhibits IgE binding to group I allergens from eight different grass species
The Journal of allergy and clinical immunology, 1994Co-Authors: Sylvia Laffer, Markus Susani, Rudolf Valenta, Susanne Vrtala, Otto Scheiner, Ronald Van Ree, Dietrich Kraft, Michael DucheˆneAbstract:Abstract Background: Grass pollens, such as pollen from timothy grass ( Phleum pratense ), represent a major cause of type I allergy. Objective: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens. Methods: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergenPhl p I. A recombinantPhl p I-β -galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli . Using recombinantPhl p V andPhl p I , we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens. IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution. Results: Preadsorption of patients' sera with recombinant nonfusionPhl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species. Conclusion: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species.
-
Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.
International archives of allergy and immunology, 1992Co-Authors: Rudolf Valenta, Susanne Vrtala, D. Kraft, C. Ebner, Otto ScheinerAbstract:In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.
Sylvia Laffer - One of the best experts on this subject based on the ideXlab platform.
-
Comparison of recombinant timothy grass pollen allergens with natural extract for diagnosis of grass pollen allergy in different populations
The Journal of Allergy and Clinical Immunology, 1996Co-Authors: Sylvia Laffer, Helmut Pairleitnera, Christian Schweiger, Hans Grönlundd, Toyota Ishii, Markus Susani, Susanne Spitzauer, Günter Menz, Gabrielle Pauli, Hendrik NolteAbstract:Abstract BACKGROUND: Complementary DNAs coding for the major timothy grass pollen (Phleum pratense) allergens Phl p 1, Phl p 2, and Phl p 5 and birch profilin were isolated, expressed as recombinant nonfusion proteins in Escherichia coli, and purified. OBJECTIVE: In this study the in vitro IgE-binding capacity of recombinant Phl p 1, Phl p 2, Phl p 5, and birch profilin and their IgE recognition frequencies were investigated by using sera from different populations. METHODS: One hundred eighty-three sera from patients allergic to grass pollen were obtained from different populations in Europe, Japan, and Canada. The sera were selected according to clinical criteria, skin testing, and RAST (CAP system; Pharmacia, Uppsala, Sweden) and then tested for IgE reactivity with natural and purified recombinant timothy grass pollen allergens by ELISA and Western blot. RESULTS: Most (94.5%) of the patients allergic to grass pollen could be diagnosed with a combination of recombinant Phl p 1, Phl p 2, Phl p 5, and profilin by means of ELISA. Sera that did not react with the recombinant allergens contained low levels of timothy grass pollen–specific IgE. Although considerable variability in IgE recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy CAP results and the combination of recombinant allergens in all 183 tested sera ( r = 0.87). CONCLUSIONS: Despite considerable variability in the IgE recognition frequency, purified recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5) and profilin permitted successful in vitro diagnosis of grass pollen allergy in 94.5% of allergic individuals from different populations. The addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity. (J ALLERGY CLIN IMMUNOL 1996;98:652-8.)
-
Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5)1
The Journal of allergy and clinical immunology, 1996Co-Authors: Susanne Vrtala, Markus Susani, Sylvia Laffer, Dietrich Kraft, Wolfgang R. Sperr, Peter Valent, Christiane Dolecek, Rudolf ValentaAbstract:Background: Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5. Objective: To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli , the cDNAs were inserted into expression plasmids. Methods: The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays. Results: Milligram amounts of pure recombinant allergens were obtained from cultured E. coli . IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils. Conclusion: Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.
-
T Cell Epitopes of Phl p 1, Major Pollen Allergen of Timothy Grass (Phleum pratense)
Advances in experimental medicine and biology, 1996Co-Authors: Siegfried Schenk, Markus Susani, Sylvia Laffer, Rudolf Valenta, Michael Duchêne, Otto Scheiner, Heimo Breiteneder, Nader Najafian, Gottfried Fischer, Dietrich KraftAbstract:Recently several group I grass pollen allergens, such as Phl p 1 from timothy grass (Phleum pratense), some isoforms of Loi p 1 from rye-grass (Lolium perenne), and Sec c 1 from rye (Secale cereale; S. Laffler, unpublished results) have been characterized by molecular cloning techniques1–3 and were found to display a high degree of amino acid (AA) sequence-similiarity. T lymphocytes play an important role in the induction of IgE-synthesis, and therefore in the development of Type I allergy4–6. In this study humoral and cellular responses to recombinant Phl p 1 (from timothy grass) were analyzed. T cell lines (TCL) and T cell clones (TCC) were established to determine T cell epitopes of this allergen. IgE-crossreactivity among pollen allergens of different grass species has been observed on the IgE-level1. We investigated T cell-crossreactivity among grass group I allergens. The knowledge of major and/or crossreacting T cell epitopes of grass pollen allergens2,7 is important for the development of vaccines for immunotherapy (IT) in the future.
-
IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens
The Journal of allergy and clinical immunology, 1994Co-Authors: Sylvia Laffer, Susanne Vrtala, Michael Duchêne, Otto Scheiner, Ronald Van Ree, Dietrich Kraft, Rudolf ValentaAbstract:Abstract A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens. In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed. Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen–specific IgE was estimated. Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I . The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens. The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen–specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy. (J ALLERGY CLIN IMMUNOL 1994;94:88-94.)
-
Complementary DNA cloning of the major allergenPhl p I from timothy grass (Phleum pratense); recombinantPhl p I inhibits IgE binding to group I allergens from eight different grass species
The Journal of allergy and clinical immunology, 1994Co-Authors: Sylvia Laffer, Markus Susani, Rudolf Valenta, Susanne Vrtala, Otto Scheiner, Ronald Van Ree, Dietrich Kraft, Michael DucheˆneAbstract:Abstract Background: Grass pollens, such as pollen from timothy grass ( Phleum pratense ), represent a major cause of type I allergy. Objective: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens. Methods: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergenPhl p I. A recombinantPhl p I-β -galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli . Using recombinantPhl p V andPhl p I , we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens. IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution. Results: Preadsorption of patients' sera with recombinant nonfusionPhl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species. Conclusion: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species.
Susanne Vrtala - One of the best experts on this subject based on the ideXlab platform.
-
Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5)1
The Journal of allergy and clinical immunology, 1996Co-Authors: Susanne Vrtala, Markus Susani, Sylvia Laffer, Dietrich Kraft, Wolfgang R. Sperr, Peter Valent, Christiane Dolecek, Rudolf ValentaAbstract:Background: Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5. Objective: To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli , the cDNAs were inserted into expression plasmids. Methods: The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays. Results: Milligram amounts of pure recombinant allergens were obtained from cultured E. coli . IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils. Conclusion: Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.
-
cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli : comparison with birch pollen profilin
Biochemical and biophysical research communications, 1994Co-Authors: Rudolf Valenta, Tanja Ball, Susanne Vrtala, Michael Duchêne, D. Kraft, Otto ScheinerAbstract:Abstract Profilin, an actin-binding protein,was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass ( Phleum pratense ) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a β-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.
-
IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens
The Journal of allergy and clinical immunology, 1994Co-Authors: Sylvia Laffer, Susanne Vrtala, Michael Duchêne, Otto Scheiner, Ronald Van Ree, Dietrich Kraft, Rudolf ValentaAbstract:Abstract A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens. In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed. Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen–specific IgE was estimated. Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I . The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens. The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen–specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy. (J ALLERGY CLIN IMMUNOL 1994;94:88-94.)
-
Complementary DNA cloning of the major allergenPhl p I from timothy grass (Phleum pratense); recombinantPhl p I inhibits IgE binding to group I allergens from eight different grass species
The Journal of allergy and clinical immunology, 1994Co-Authors: Sylvia Laffer, Markus Susani, Rudolf Valenta, Susanne Vrtala, Otto Scheiner, Ronald Van Ree, Dietrich Kraft, Michael DucheˆneAbstract:Abstract Background: Grass pollens, such as pollen from timothy grass ( Phleum pratense ), represent a major cause of type I allergy. Objective: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens. Methods: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergenPhl p I. A recombinantPhl p I-β -galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli . Using recombinantPhl p V andPhl p I , we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens. IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution. Results: Preadsorption of patients' sera with recombinant nonfusionPhl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species. Conclusion: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species.
-
cdna cloning of a major allergen from timothy grass phleum pratense pollen characterization of the recombinant phl pv allergen
Journal of Immunology, 1993Co-Authors: Susanne Vrtala, Sylvia Laffer, Wolfgang R. Sperr, Peter Valent, W.-d. Müller, I Reimitzer, K Lechner, H Rumpold, D. KraftAbstract:We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found to other major grass pollen allergens from Kentucky bluegrass (Poa pratense) as well as from rye grass (Lolium perenne) which originally were believed to form different identities. Using different monoclonal and polyclonal antibodies raised against group V allergens we identified the recombinant protein as a group V allergen from timothy grass, Phl p V. In IgE-binding studies it is demonstrated that the rPhl p V allergen can be used to block binding of patients' IgE to natural group V isoallergens on two-dimensional immunoblots. IgE inhibition experiments show that up to 60% of grass pollenspecific IgE can be preadsorbed with the rPhl p V allergen from patients sera. The purified rPhl p V induced specific histamine release of blood basophils from grass pollen-allergic patients. This emphasizes the usefulness of the rPhl p V for diagnostic and therapeutic purposes and corroborates the view that specific diagnosis and therapy of type l allergy could be performed with a limited panel of relevant recombinant allergens.
D. Kraft - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of Phl p 4, a major timothy grass (Phleum pratense) pollen allergen
The Journal of allergy and clinical immunology, 1996Co-Authors: Sabine Fischer, D. Kraft, Monika Grote, B. Fahlbusch, W.-d. Müller, Rudolf ValentaAbstract:Abstract Background: Group 4 grass pollen allergens represent glycoproteins with a molecular weight of 50 to 60 kd, which are present in many grass species. Almost 75% of patients allergic to grass pollen display IgE reactivity to group 4 allergens, which hence can be regarded as major grass pollen allergens. Objective: In this study attempts were made to obtain information regarding the immunologic properties, localization, and occurrence of Phl p 4 and related allergens. Methods: Phl p 4 was detected in timothy grass pollen extracts by immunoblotting with serum IgE and monoclonal antibodies and was localized in pollen by immunelectron microscopy. A peptide sequence from Phl p 4 was obtained by amino acid sequencing. The resistance of Phl p 4 against trypsin was analyzed after trypsin treatment of timothy grass pollen extracts with serum IgE and monoclonal antibodies. Cross-reactivities between Phl p 4 and Amb a 1, the major allergen of ragweed, were studied by using monoclonal antibodies and by IgE-inhibition studies. Results: Phl p 4 was characterized as a trypsin-resistant major timothy grass pollen allergen. By immunelectron microscopy Phl p 4 was localized in the exine, cytoplasm, and amyloplast of timothy grass pollen. Significant sequence similarities of a Phl p 4 10 amino acid peptide with Amb a 1, the major ragweed allergen, could be found. The immunologic similarity of Phl p 4 and Amb a 1 was confirmed by cross-reactivity of monoclonal antibodies and patients' IgE. Conclusion: Phl p 4 represents a trypsin-resistant major timothy grass pollen allergen with immunologic similarities to the major ragweed allergen Amb a 1 and therefore must be considered an important cross-reactive component in grass pollen and weed pollen allergy. (J ALLERGY CLIN IMMUNOL 1996;98:189-98.)
-
cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli : comparison with birch pollen profilin
Biochemical and biophysical research communications, 1994Co-Authors: Rudolf Valenta, Tanja Ball, Susanne Vrtala, Michael Duchêne, D. Kraft, Otto ScheinerAbstract:Abstract Profilin, an actin-binding protein,was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass ( Phleum pratense ) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a β-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.
-
cdna cloning of a major allergen from timothy grass phleum pratense pollen characterization of the recombinant phl pv allergen
Journal of Immunology, 1993Co-Authors: Susanne Vrtala, Sylvia Laffer, Wolfgang R. Sperr, Peter Valent, W.-d. Müller, I Reimitzer, K Lechner, H Rumpold, D. KraftAbstract:We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found to other major grass pollen allergens from Kentucky bluegrass (Poa pratense) as well as from rye grass (Lolium perenne) which originally were believed to form different identities. Using different monoclonal and polyclonal antibodies raised against group V allergens we identified the recombinant protein as a group V allergen from timothy grass, Phl p V. In IgE-binding studies it is demonstrated that the rPhl p V allergen can be used to block binding of patients' IgE to natural group V isoallergens on two-dimensional immunoblots. IgE inhibition experiments show that up to 60% of grass pollenspecific IgE can be preadsorbed with the rPhl p V allergen from patients sera. The purified rPhl p V induced specific histamine release of blood basophils from grass pollen-allergic patients. This emphasizes the usefulness of the rPhl p V for diagnostic and therapeutic purposes and corroborates the view that specific diagnosis and therapy of type l allergy could be performed with a limited panel of relevant recombinant allergens.
-
Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.
International archives of allergy and immunology, 1992Co-Authors: Rudolf Valenta, Susanne Vrtala, D. Kraft, C. Ebner, Otto ScheinerAbstract:In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.