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S Laffer

Publications and source records attributed to S Laffer.

27 records · Page 2Linked to original sources
PubMed1994

Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species.

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 allergen Phl p I. A recombinant Phl p I-beta-galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli. Using recombinant Phl p V and Phl 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 nonfusion Phl 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.

Allergens↗
PubMed1994

B-cell epitopes of allergens determined by recombinant techniques; use for diagnosis and therapy of type I allergy.

In the present manuscript, the immunological and functional in vitro properties of recombinant plant allergens are summarized. Recombinant tree pollen allergens (major birch pollen allergen-BetvI, birch profilin-BetvII) and recombinant timothy grass pollen allergens (PhlpI, PhlpV, and PhlpII) were compared with the natural counterparts regarding IgE-binding properties and capacity to release histamine from patients' basophils. In addition, experimental in vivo models of Type I allergy, based on recombinant allergens, are discussed. The major conclusion is that recombinant allergens can be seriously considered as candidates for diagnosis of Type I allergy allowing to establish specific allergograms for the individual patients. The in vivo data obtained in mouse and primate systems indicate that recombinant allergens can be used to set up close-to-man models of Type I allergy. Such in vivo models are useful to test the effects of already established therapeutic approaches and also allow to develop therapeutical concepts which are based on the use of recombinant allergens. Examples of specific therapeutical concepts are presented.

Allergens↗
PubMed1993

Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen.

Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C-terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE-binding capacity as the natural allergen.

Allergens↗
PubMed1993

cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen.

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.

Amino Acid Sequence↗
PubMed1993

[New concepts in therapy of type I allergic diseases].

The molecular characterization of allergens with recombinant DNA techniques allowed cDNA sequences to be obtained and, hence, information regarding the primary structure of allergens. It is now possible to express well-defined recombinant allergens in heterologous expression systems and to obtain large amounts of highly pure recombinant allergens for the improvement of current diagnosis and therapy of type I allergic diseases. Due to extensive cross-reactivities and structural similarities of the relevant allergens it is possible to define a limited number of allergens, which is a prerequisite for allergen-specific therapeutic concepts. Specific concepts of active immunotherapy and strategies of passive therapeutic interference based on recombinant techniques are discussed.

Allergens↗
PubMedDate not supplied

Common IgE-epitopes of recombinant Phl p I, the major timothy grass pollen allergen and natural group I grass pollen isoallergens.

Grass pollen allergens are potent elicitors of Type I allergy. More than 95% of grass pollen allergic patients display IgE-cross-reactivity to group I grass pollen allergens of different grass species. A cDNA coding for the major timothy grass pollen allergen, Phl p I, was isolated previously. To investigate the presence of common IgE-epitopes among naturally occurring group I grass pollen isoallergens, Phl p I was expressed in Escherichia coli and used for IgE-absorption experiments. Recombinant Phl p I was able to inhibit IgE-binding to most of group I isoallergens from seven grass species as identified by two dimensional electrophoresis. When tested in competitive ELISA experiments, recombinant Phl p I bound a high percentage of grass pollen specific IgE. The results indicate that recombinant Phl p I shares many of the IgE-epitopes with natural group I grass pollen allergens and hence may represent a useful tool for specific diagnosis and therapy of grass pollen allergy.

Allergens↗
PubMedDate not supplied

An in vitro model for the allergen-IgE-FcARI interaction.

BACKGROUND: The interaction of immune complexes consisting of allergens and allergen-specific IgE with the high-affinity Fcepsilon receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate-type symptoms result from the release of biological mediators due to allergen-induced cross-linking of FcepsilonRI receptors on mast cells and basophils, whereas FcepsilonRI-mediated presentation of allergen-IgE complexes may contribute to late-phase symptoms through enhanced T cell activation. The interaction of allergens/allergen-specific IgE/FcepsilonRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. METHODS AND RESULTS: A molecular model of the allergen-IgE-FcepsilonRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus-expressed purified human alpha chain of FcepsilonRI. The chimeric Bet v 1-specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose-immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse-derived Bip 1. Bound chimeric Bip 1 was detected with 125I-labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE-alpha chain interaction. CONCLUSIONS: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.

Allergens↗