Biomedical subjects

D Kraft

Publications and source records attributed to D Kraft.

At least 181 records · Page 10Linked to original sources

PubMed1994

Molecular and functional characterization of allergens: basic and practical aspects.

Well-defined allergen preparations are, in the first line, a prerequisite for exact diagnosis, but will be supposedly useful tools in immunotherapy of Type I (IgE-mediated) allergic diseases. The allergens have to be available in standardized and highly purified form in sufficient quantities. By applying recombinant DNA techniques this goal can be achieved with respect to both, characterization and reproducibility of allergen preparations. As an example, purified recombinant non-fusion Betv1 revealed identical immunological properties with respect to interaction with both, anti-Betv1 antibodies and Betv1-specific T cell clones when compared with natural Betv1 purified from birch pollen. Moreover, cloning of allergens yielded a number of deduced primary structures of allergens, which allows computer-aided comparisons with already known amino acid sequences. Significant sequence similarities with well-described proteins may point at a biological and biochemical function of the cloned allergen, which might be of interest for considerations why a certain protein within an extract represents an allergen. Furthermore, the interaction of small peptides synthesized according to amino acid sequences of cloned allergens with allergen-specific T and B cells can be investigated. Respective results will yield information about the regulation of IgE synthesis and, thus, might point at new concepts of immunotherapy.

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

Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

Pollens from trees of the order Fagales (e.g. birch, alder, hazel, and hornbeam) all contain one major allergen--the main cause for tree pollen allergy. So far the major allergens from birch (Bet v 1), alder (Aln g 1), hazel (Cor a 1), and hornbeam (Car b 1) have been characterized, showing high sequence similarity with each other (1-4). We present the molecular and immunologic characterization of Cas s 1, the major allergen from the European chestnut (Castanea sativa). From aqueous pollen extracts from European chestnut a protein was purified to homogeneity and was subjected to further investigation. The protein revealed a Mr of 22 kDa and was shown to represent the major allergen of the European chestnut (immunoblotting, histamine release) and designated Cas s 1. Despite a marked difference in Mr, Cas s 1 shows significant amino acid sequence similarity at the N-terminus and is antigenically closely related to the major birch pollen allergen Bet v 1 (17 kDa), as shown by binding to the anti-Bet v 1 monoclonal antibody BIP-1 and by IgE-inhibition tests using recombinant Bet v 1.

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

Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-binding properties.

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding to these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3'-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3'-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96-99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71-73% (80.5-83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Car b I and isoforms, the major allergen from hornbeam, 75.5-76.7% identity (83.6-85% similarity) and 83.6-89.9% sequence identity (89.3-95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor a I. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.

Allergens↗

PubMed1993

Identification of multiple T cell epitopes on Bet v I, the major birch pollen allergen, using specific T cell clones and overlapping peptides.

Eleven T cell clones (TCC) with specificity for Bet v I were established from the peripheral blood of six birch pollen allergic donors. Bet v I is the major allergen of birch (Betula verrucosa) pollen and shows high homology to the major allergens of pollens of other trees within the order fagales (hazel, alder, hornbeam, oak, etc.), which represent important inhalant allergens in the northern hemisphere. The TCC were shown to react with purified natural, as well as with purified recombinant Bet v I. All clones showed the helper cell phenotype (CD3+CD4+) and expressed the TCR-alpha/beta. The cytokine production pattern in response to stimulation with allergen resulted in enhanced production of IL-4 in 9 of 11 clones. The clones were used for T cell epitope mapping on the Bet v I molecule. For this purpose, peptides with a length of 12 amino acids each and overlapping for 10 residues were synthesized following the amino acid sequence of Bet v I. These 75 peptides were used to stimulate Bet v I-specific T cell clones. Our experiments revealed 7 distinct T cell epitopes on the Bet v I molecule. The epitopes were scattered over the whole molecule, 2 sequences were in agreement with an algorithm previously described for the prediction of T cell epitopes. In 3 cases, we could identify distinct TCC specificities within single individuals. Furthermore, for each donor, none of the peptides representing epitopes for TCC inhibited the binding of IgE antibodies to Bet v I. These results suggest that T cells and IgE antibodies from the same individual recognize different structures on the Bet v I allergen.

Allergens↗

PubMed1993

Multiple T cell specificities for Bet v I, the major birch pollen allergen, within single individuals. Studies using specific T cell clones and overlapping peptides.

Twenty-five T cell clones specific for Bet v I were established from the peripheral blood of two birch pollen-allergic patients. The T cell epitopes of these clones were mapped using dodecapeptides overlapping for 2 amino acids (neighbors share 10 residues) spanning the whole amino acid sequence of the protein (159 amino acids). In total, 7 epitopes could be detected. One donor displayed 6 distinct T cell specificities for the Bet v I molecule in 14 T cell clones; for the other donor, 4 stimulating peptides for 11 clones could be identified. Two T cell epitopes were recognized by both subjects. One of these might represent an immunodominant epitope located at amino acid position 77-92 of the Bet v I molecule, as in 13/25 T cell clones activation could be induced by this amino acid sequence. One T cell clone reacted with purified pollen-derived Bet v I, but neither with any peptide synthesized according to a Bet v I-encoding cDNA nor with the respective recombinant protein. Upon stimulation with allergen, the majority of the clones (21/24) revealed the TH0 or TH2 type of cytokine production (interleukin-4 production), indicating their importance in the pathogenesis of the allergic disease.

Allergens↗

PubMed1993

The expression and cellular distribution of adhesion molecules CD2/LFA-3 and ICAM-1/LFA-1 on mononuclear cells in squamous cell carcinoma of the head and neck.

The expressions and cellular distributions of two pairs of adhesion molecules CD2/LFA-3 (leukocyte function-associated antigen-3) and LFA-1/ICAM-1 (intercellular adhesion molecule-1) were examined in inflammatory cellular infiltrates of advanced squamous cell carcinomas of the head and neck by immunohistochemical techniques including double-staining methods. Thirteen patients were investigated using the following monoclonal antibodies (mAbs): CD2, LFA-3 (CD58), ICAM-1 (CD54), LFA-1 (CD11a), the alpha/beta and gamma/delta T-cell receptor, pan T cells and broadly distributed monocyte/macrophage (m/m phi) [Fc gamma RII (CD32), 25F9, RM3/1]. LFA-3 staining was observed on a high number of cells (968 +/- 112 cells/mm2), correlating to the number of Fc gamma RII (CD32; P < 0.01), 25F9 (P < 0.05) and RM3/1 (P < 0.05) positive m/m phi. Its ligand CD2 was found on 365 +/- 126 cells/mm2, representing about 50% of CD3+ cells (730 +/- 286 cells/mm2). CD2 positivity correlated to CD3 and CD8 (P < 0.01) but not to CD4+ T cells. LFA-1 and ICAM-1 were expressed on lymphocytes as well as on m/m phi. ICAM-1+ cells (902 +/- 205 cells/mm2) correlated to CD3+, CD8+ and RM3/1+ cells (P < 0.01). LFA-1 positivity (803 +/- 255 cells/mm2) showed correlations to nearly all investigated antigens, as well as to CD4+ T cells (P < 0.05). These results show that different m/m phi subsets display distinct patterns of adhesion molecule expressions suggesting different pathways of regulation. The CD3+ lymphocyte population revealed a lack of CD2 expression that was more pronounced in the CD4+ subset and indicated impaired lymphocyte function.

Adult↗

PubMed1993

Hydroxyethylstarch deposits in human skin--a model for pruritus?

Severe itching for unknown reasons has been reported after administration of hydroxyethylstarch (HES) in haemodilution therapy of humans. After HES treatment, vacuoles in cells of various organs in humans have been shown, predominantly affecting the mononuclear phagocyte system. These vacuoles present indirect evidence for phagocytosis of HES particles. Since phagocytosis is also known to occur in the skin, this organ might represent a target for HES deposition, resulting in subsequent release of mediators responsible for the observed itching. The aim of the present investigation was to study skin biopsies of patients, who had received HES and suffered subsequently from itch. Skin sections were investigated for morphological impairment by means of light and electron microscopy, immunohistochemistry and immunoelectron microscopy using a polyclonal anti-HES antiserum. Storage of HES was demonstrated in the skin of all patients, mainly in dermal macrophages, endothelial cells of blood and lymph vessels, some perineural cells and endoneural macrophages of larger nerve fascicles, some keratinocytes and Langerhans cells. Treatment with antihistaminic agents proved ineffective in these patients; this fits with the observation that morphological signs of histamine release from mast cells were absent. These findings indicate that other mediators from HES-affected cells must be responsible for the development of the itching. Thus, investigation of HES storage may be a useful contribution to the elucidation of release of itch mediators and induction of pruritus.

Adult↗

PubMed1993

Human monocytes and keratinocytes in culture ingest hydroxyethylstarch.

Severe itching for unknown reasons has been reported after administration of hydroxyethylstarch (HES) in haemodilution therapy of humans. In skin sections from patients who had received HES for otological reasons HES deposition was found by immunohistochemical methods in keratinocytes, epidermal dendritic cells, endothelial cells and macrophages. The aim of the present investigation was to study the interaction of cultured human monocytes and keratinocytes, normal (HNK) and malignant (KB, A431), with FITC-conjugated HES (200/0.5). Peripheral blood monocytes from healthy donors were isolated by Ficoll-Hypaque gradient centrifugation and adherence to plastic surfaces. Cells were incubated with HES-FITC (1 mg/ml) for 10, 20, 30, 60 and 120 min at 37 degrees C, washed several times to minimize cell-associated HES-FITC. The uptake of HES-FITC (200/0.5) was detected by fluorescence microscopy and flow cytometry. Monocytes and keratinocytes (HNK, KB, A431) acquired significant amounts of HES-FITC after 120 min. Endocytosis was blunted by the addition of unconjugated HES in ten-fold concentration. At 4 degrees C, a further inhibition was achieved, indicating an uptake of HES-FITC by monocytes and keratinocytes in vitro. These results indicate that a connection between the storage of HES by keratinocytes and monocytes and the observed itching in patients might be possible.

Cells, Cultured↗

PubMed1993

Induction of specific histamine release from basophils with purified natural and recombinant birch pollen allergens.

As much as 15% of the population in industrialized countries suffers from type I allergic symptoms (rhinitis, conjunctivitis, and bronchial asthma). One approach toward this disease involves the production of recombinant allergens in Escherichia coli and their purification for diagnostic and therapeutic purposes. In this study we compared the IgE-binding capacity of natural and recombinant birth allergens with their functional ability to release histamine from allergic patients' basophils via cross-linking of high-affinity Fc epsilon-receptors. The recombinant as well as pollen-derived Bet v I and birch profilin (Bet v II) were purified and tested in parallel on IgE immunoblots and in in vitro histamine release tests (n = 21). We observed an excellent correlation between allergen-induced histamine release and birch pollen RAST (r = 0.881, p < 0.002). Nonspecific histamine release as a result of a cell toxic effect of the allergen preparations was never observed from basophils of donors without birch pollen allergy. The specificity of the presented effector model is also documented by the specific desensitization of patients' basophils with the recombinant allergens. These data may provide a basis for the use of purified recombinant allergens in sensitive and specific in vitro allergy tests monitoring the effector situation in allergic patients, which therefore may represent a possible alternative for in vivo diagnostic methods.

Allergens↗

PubMed1993

Allergens from birch pollen and pollen of the European chestnut share common epitopes.

Type I allergy to pollen of the European chestnut (Castanea sativa) represents a major cause of pollinosis in (sub) Mediterranean areas. Using sera from 14 patients with established allergy to pollen of the European chestnut, 13/14 sera (92%) showed IgE-binding to a 22 kD protein, 2/14 (14%) displayed additional binding to a 14 kD protein and 1/14 (7%) bound only to the 14 kD protein of European chestnut pollen extract. Two monoclonal mouse antibodies, BIP 1 and BIP 4, directed against different epitopes of Bet v I (the major birch pollen allergen), and a rabbit antibody to recombinant birch profilin (rBet v II) were used to characterize the proteins of the European chestnut pollen. The recombinant birch pollen allergens, rBet v I and rBet v II (profilin) were employed to show common allergenic structures on proteins from both birch and European chestnut pollen by IgE-inhibition experiments. Despite the fact that the 22 kD protein displayed a higher molecular weight in comparison to the 17 kD major birch pollen allergen, Bet v I, we could demonstrate reactivity of both monoclonal antibodies, BIP 1 and BIP 4, with this protein. A complete inhibiton of IgE-binding to this 22 kD protein was shown by pre-incubating sera with purified recombinant Bet v I. In addition, the 14 kD protein could be identified by IgE-inhibition studies with recombinant Bet v II and by using a rabbit anti-profilin antibody as the profilin from pollen of the European chestnut.

Allergens↗

PubMed1993

Characterisation of dog allergens by means of immunoblotting.

Sera from 75 patients with clinical type I allergy against dogs were investigated by means of immunoblotting using extracts prepared from dog hair/dander (CAN XI D) and saliva. In addition, selected sera were tested on extracts made of hair, skin, salivary glands (parotis and submandibularis), serum and liver. A 69-kD IgE-binding protein was identified in all extracts tested with an incidence of approximately 40% and shown to be dog albumin by means of inhibition experiments. In 96% of patients' sera IgE antibodies reactive with a 19-kD and/or a 23-kD protein of the hair/dander extract (CAN XI D) were observed. IgE binding to a 23-kD band was also detected in the hair and saliva extracts, but not in skin, salivary gland, serum and liver extracts. A 19-kD IgE-binding protein was strongly expressed in skin and to a lesser degree in saliva, but not in hair, serum and liver. Preincubation of patients sera with the hair extract and subsequent probing with the hair/dander extract (CAN XI D) inhibited IgE binding to the 23 kD protein whereas preincubation with the skin extract abolished IgE binding to the 19-kD protein. Using the hair/dander extract as inhibitor, IgE binding to the 19- and 23-kD proteins of saliva was abrogated. Thus it is concluded that the 23-kD protein is preferentially expressed in hair and saliva whereas the 19-kD protein is found in saliva and skin. Furthermore these two proteins are likely to represent immunologically independent major allergens.

Adolescent↗

PubMed1993

Detection of IgE antibodies specific for allergens in cow milk and cow dander.

Sera from patients with cow milk protein allergy (n = 6) and cow dander allergy (n = 5) were analyzed for reactivity of IgE antibodies specific for allergens derived from milk and cow dander. The cow milk- and cow dander-allergic patients exhibited elevated specific IgE levels to milk and to cow dander, respectively, as determined by RAST. IgE immunoblot analysis revealed that cow milk-allergic patients exhibited IgE binding to the milk allergens casein, beta-lactoglobulin, alpha-lactalbumin and bovine serum albumin. Four of six cow milk-reactive patients also exhibited IgE binding to cow dander proteins with molecular weights of 20, 22, 36, 50 and > 200 kD. IgE from the sera of cow dander-allergic patients reacted with the major allergens of cow dander (20 and 22 kD) and with other proteins with molecular weights of 24, 36, 42, 50 and 70 kD. Only one out of five of these sera displayed IgE reactivity with cow milk proteins with molecular weights of 69, 92 and > 200 kD. Inhibition studies revealed the cross-reactive nature of the IgE antibodies. Preincubation of cow milk-positive sera with cow milk and subsequent immunoblotting led to complete blocking of IgE binding to cow dander proteins and to cow milk proteins. Preincubation of cow milk-positive sera with cow dander extract led to blocking of IgE binding only to defined cow milk proteins: these could be identified in 2 cases as casein and in 1 case as beta-lactoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1993

Properties of tree and grass pollen allergens: reinvestigation of the linkage between solubility and allergenicity.

In this study we reinvestigated the kinetics of allergen release from birch pollen (Betula verrucosa) and timothy grass pollen (Phleum pratense) using different protein extraction procedures, immunoblotting with specific antibodies and immune electron microscopy. Pollen allergens such as the major birch pollen allergen, Bet v I, the major timothy grass pollen allergens, Phl p I and Phl p V, group-II/III allergens from timothy grass and profilins were released rapidly and in large amounts from hydrated pollen. Within a few minutes pollen allergens could be detected in aqueous supernatants prepared from birch and grass pollen with serum IgE or specific antibodies. In parallel the allergen content in the pollen pellet fractions decreased. A nonallergenic protein such as heat shock protein 70 can be extracted in sufficient amounts only with harsh extraction procedures. Immune electron microscopy of dry and rehydrated birch pollens showed that after short hydration, the major birch pollen allergen, Bet v I, migrated into the exine and to the surface of intact pollen grains, whereas profilin, against which a lower percentage of patients is sensitized, was retained in the pollen grain. Comparing the amino acid composition and hydrophilicity of the tested allergens with a nonallergenic protein such as heat shock protein 70, no significant difference was noted. In agreement with earlier observations we conclude that the allergenic properties of proteins are rather linked to the amount and speed of solubility from airborne particles than to intrinsic properties.

Allergens↗

PubMed1993

[Effector cells in allergy: biological principles and new pharmacologic concepts].

The clinical symptoms of allergy are caused by cellular (IgE-triggered) responses to an allergen. Effector cells of allergy include eosinophil and basophil granulocytes, as well as tissue mast cells. Growth and accumulation, as well as IgE-dependent and independent functions of these cells are regulated by distinct proteohormones and peptides. The hemopoietic cytokines IL-3 (interleukin-3), IL-5 and GM-CSF (granulocyte-macrophage colony-stimulating factor) are involved in the regulation of basophils (and eosinophils), whereas the ligand for c-kit, SCF (stem cell factor) is a mast cell-specific agonist. Basophils and mast cells express high-affinity IgE-binding sites. Allergen binding to IgE on mast cells and basophils, and consecutive cross-linking of IgE receptors is followed by production and/or secretion of inflammatory mediator substances. Specific activation and deactivation of mast cells/basophils in vitro has been demonstrated by use of recombinant cytokines and allergens, and specific haptens or by use of novel drugs, and should lead to epitope-specific diagnosis and better management of allergic diseases in the future.

Allergens↗

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↗