Biomedical subjects

W M Becker

Publications and source records attributed to W M Becker.

At least 55 records · Page 3Linked to original sources

PubMed1995

Structural investigations of the major allergen Phl p I on the complementary DNA and protein level.

Until now investigations of group I grass allergens have mainly been performed on ryegrass allergen (Lol p I). We studied this major allergen grass group with timothy grass pollen (Phl p I), a very common and important cause of type I allergy, to determine intraspecific and interspecific variations among different grass species. By immunoscreening a timothy grass pollen complementary DNA library we obtained three full-length clones. They revealed identical nucleotide sequences in the coding regions consisting of 262 amino acids, including a leader sequence of 23 amino acid residues. The comparison of our data with the amino acid sequences deduced from Lol p I and Hol 1 I clones showed sequence identities of greater than 85% and homologies of greater than 90%, indicating a high degree of sequence conservation. Despite the high degree of homology, amino acid differences were in immunodominant positions, which may be responsible for the differing immune response to group I allergens of different grass species.

Allergens↗

PubMed1995

Characterization of the allergen group VI in timothy grass pollen (Phl p 6). I. Immunological and biochemical studies.

For the identification and characterization of allergen grass group VI we performed two-dimensional immunoblotting of timothy grass pollen (Phleum pratense). Two intense 13-kD protein spots of pI 5.2 and 5.5 were found to be IgE reactive. By N-terminal microsequencing and amino acid analysis we identified them as Phl p 6 isoallergens. An antiserum was raised against Phl p 6 by immunizing BALB/c mice with allergen bearing nitrocellulose particles of one isoform. The antiserum revealed an intense reactivity to Phl p 6 isoforms, but also showed a weak cross-reactivity with Phl p 5 allergens. After immunoabsorption of patients' serum to Phl p 6 spots on the blotting membrane, we were able to demonstrate that the eluted human IgE antibodies cross-react with the grass group V allergens as well. Therefore, Phl p 5 and Phl p 6 possess one or more common IgE binding epitopes.

Allergens↗

PubMed1995

Characterization of the allergen group VI in timothy grass pollen (Phl p 6). II. cDNA cloning of Phl p 6 and structural comparison to grass group V.

The complete primary structure of the major allergen Phl p 6 was determined by cDNA cloning. A cDNA library of timothy grass pollen was screened by a Phl p 6 directed mouse antiserum. Six clones were obtained. From the cDNA data we deduced a protein sequence of 110 amino acids. While the size of the leader sequences varied considerably between the different clones, only one amino acid exchange affected the mature proteins. Sequence comparison of group VI and V allergens showed a high degree of homology in the N- and C-terminal regions. The structural data strongly indicate that group VI and V are independent groups of allergens, which have derived from a common precursor gene.

Allergens↗

PubMed1995

[Immunopathogenesis of extrinsic allergic alveolitis].

Inhalation of a variety of organic dusts may cause the onset of hypersensitivity pneumonitis (HP) finally leading to irreversible pulmonary fibrosis in some individuals. So far, the pathogenesis of HP remains partially unclear. Besides patient-related factors this is probably attributable to the complex composition of the causative dusts: in addition to specific antigens that may induce type III and type IV reactions they contain a variety of additional components like particles and toxins with the ability to promote several antigen-independent reactions. During an acute episode of HP a marked alveolitis dominated by polymorphonuclear cells develops. As we showed these polymorphonuclear cells are in an activated state and may therefore cause pronounced damage in the lung interstitium. Based on these and other findings we believe that polymorphonuclear cells are of predominant importance for the pathogenesis of HP.

Acute Disease↗

PubMed1994

Major allergen Phl p Va (timothy grass) bears at least two different IgE-reactive epitopes.

It is established that most grass pollen allergens consist of several isoforms of which the function is mainly still unknown. A number of these allergens belonging to group V have been cloned, sequenced, and expressed. Antigenic sites and IgE-reactive epitopes of the major allergen Phl p Va, are unknown. We have identified the complete cDNA sequence of a Phl p Va isoallergen by immunoscreening of a timothy grass pollen cDNA library and mixed oligonucleotide primed amplification of N-terminal cDNA. Additionally, we found an incomplete isoallergenic cDNA clone of the same protein. Immunoreactivity of the fusion proteins with patients' sera and monoclonal antibodies showed that the clones represent group Va allergens. Comparison of deduced amino acid sequences with published sequences of Lol p V and Poa p IX revealed a homology of 81.1% and 86.9%, respectively. With affinity-purified IgE antibodies recognizing the recombinant fusion protein, we can demonstrate the existence of a common group V IgE-reactive epitope. By construction of both an N-terminal and a C-terminal peptide of the complete Phl p Va and cross-inhibition, we identified at least two different IgE epitopes. Eleven patients showed variable IgE immunoreactivities to both IgE-reactive epitopes.

Allergens↗

PubMed1994

Occupational sensitization to Plasmopara viticola.

Molds of the class of Oomycetes are of allergologic importance in special cases. However, probes are not commercially available for diagnostic purposes. Our case report is based on the medical history of a greenhouse worker who had atopic syndrome. He handled pure cultures of defined fungi and plants. A sensitization to pseudo mildew growing on grapevine (Plasmopara viticola) was found. Skin prick test and histamine release test results were positive when extract of P. viticola was used. Detection of IgE reactivities against pseudo mildew via binding tests, Western blot analysis, and isoelectric focusing immunoblot confirmed the diagnosis. To our knowledge these results demonstrate for the second time a sensitization to Oomycetes.

Adult↗

PubMed1994

Analysis of B-cell epitopes in the N-terminal region of Chi t I component III using monoclonal antibodies.

The hemoglobins of the midge Chironomus thummi thummi (Chi t I) are known to cause immediate-type hypersensitivity reactions in humans. Further knowledge of the antigenic sites of such allergens will provide new therapeutic approaches. The aim of our study was to identify and characterize linear B-cell epitopes of the hemoglobin component III of Chi t I (136 amino acid residues). Using the antigenic index algorithm of Jameson and Wolf (Jameson and Wolf (1988) Comput. Appl. Biosci. 4, 181-186), three linear binding sequences of this allergen molecule were predicted. Two mouse monoclonal antibodies (mAbs 3 and 6) raised against purified Chi t I component III were investigated by ELISA for their binding to nine synthetic peptides 19-21 residues in length, covering nearly the whole sequence of component III. MAb 6 recognized only one peptide (11-30) while mAb 3 bound to both N-terminal peptides (1-19 and 11-30), suggesting that the antibody binding site is located in the overlapping region. This assumption could be confirmed in ELISA with solid phase-bound recombinant peptides (RP) as well as in inhibition studies with free tryptic peptides indicating that identification of these linear B-cell epitopes is neither influenced by the method of peptide production nor by the kind of used immunoassay. To define the essential amino acid residues we investigated mAbs with solid phase-bound overlapping octamers. In the case of mAb 3, amino acids experimentally identified as essential for antibody binding (aa 13-17) are identical with those residues predicted as a B-cell epitope with the antigenic index of Jameson and Wolf.

Allergens↗

PubMed1994

Epitope analysis of isoforms of the major allergen Phl p V by fingerprinting and microsequencing.

The major allergen of timothy grass pollen (Phleum pratense), designated as Phl p V, consists of isoallergenic components of 38 and 32 kDa with pl values of 5.2-7.5 and 4.8-5.9, respectively. The different-sized proteins reveal similarities in IgE reactivity, N-terminal sequence and protein staining. For epitope analysis of these allergens a combination of enzymatic cleavage of electrophoretically separated proteins and immunoblotting techniques with subsequent N-terminal sequencing was performed. After isolation of the components from two-dimensional PAGE gels, proteins were enzymatically cleaved and separated by SDS-PAGE. By endoproteinase Glu-C cleavage six IgE-reactive fragments of each 32 kDa protein and three of each 38 kDa allergen were obtained. Microsequencing of the fragments revealed internal sequences that did not show any similarities between the different-sized allergens. Therefore, we assume only slight structural variations among allergens of similar sizes, whereas the 32 and 38 kDa proteins reveal great differences.

Allergens↗

PubMed1994

Analysis of IgG subclass and IgE response in allergic disease caused by Aspergillus fumigatus by immunoblotting techniques.

Analysis of sera from patients suffering from allergic asthma, extrinsic allergic alveolitis (EAA) due to Aspergillus fumigatus or allergic bronchopulmonary aspergillosis (ABPA) by means of IEF immunoprint and Western blot showed characteristic IgG subclasses and IgE responses. Asthmatic patients revealed specific IgE antibodies. IgG subclass reactivities in asthmatics and healthy blood donors were weak and uncharacteristic. Major allergens recognized by asthmatic patients have isoelectric points (pI) of pI 4.4 and 4.8 and molecular weights of 20, 29, 34, 38 and 45 kD. Patients suffering from EAA typically demonstrated strong IgG subclass reactivities with an emphasis to IgG1 and IgG2, while no specific IgE was found. Various major antigens could be identified. Reaction to a pI 6.0 antigen seemed to be characteristic for EAA. ABPA patients showed strong reactivities of all IgG subclasses and of IgE. An intense IgG4 reaction appeared as a typical marker for ABPA. Several major antigens and allergens could be determined. Especially the IgG4 reactivities to proteins with pI 4.6, 5.0, 5.2, 5.4 and 6.5 and molecular weights of 14, 20, 29, 34, 38 and 45 kD seemed to be a characteristic pattern in ABPA. Furthermore, IgE antibodies to culture filtrate components of 34 and 38 kD appeared to be typical in ABPA.

Alveolitis, Extrinsic Allergic↗

PubMed1994

[Molecular characterization of allergens].

Allergenic source material consists of a great number of single components of which only a small number is IgE-reactive. Since allergenic source material is the basis of diagnostics and immunotherapy, there is a strong interest in identification, characterization and quantification of allergens. A further reason of interest is to explain the pathomechanism of the type I allergy. The structure of allergens is of central importance, because only a small number of antigens are allergens and the structure of these components is essential in giving rise to symptoms. The process and further prospects of molecular characterization of allergens are described and grass pollen allergens are used as paradigm. Allergies are an excellent model to study the meaning of specificity in immunologic disorders.

Allergens↗

PubMed1994

Differences in IL-4 release by PBMC are related with heterogeneity of atopy.

Atopy is heterogeneous and the IgE immune response of patients allergic to a single allergen (monosensitized) differs from that of those allergic to multiple allergens (polysensitized). Since interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) regulate human IgE synthesis in vitro, we determined whether cytokines may be involved in the heterogeneity of atopy by comparing the serum IgE and sCD23 titres to the cytokine profile of T lymphocytes from 44 atopic patients (13 mono- and 31 polysensitized) and seven non-atopic subjects. Monosensitized patients were allergic to grass or cypress pollens or house dust mites, and polysensitized ones to many pollen species (n = 5) or many allergens (n = 26). Total serum IgE was lower in the control group than in both atopic groups and in the monosensitized group than in the polysensitized one. IgE immunoblots to orchard grass pollen and house dust mites were less heterogeneous in the monosensitized group than in the polysensitized one. IL-4 production by in vitro-activated peripheral blood mononuclear cells (PBMC) was significantly higher in the polysensitized group than in the monosensitized, and marginal in the control group. In contrast, IFN-gamma production was strongly reduced in both atopic groups, and IL-2 production comparable in the three groups. IgE and soluble CD23 (sCD23) release was higher in the atopic groups than in the control, and higher in the polysensitized group than in the monosensitized one. This study shows that PBMC of mono- and polysensitized subjects have a different IL-4 and sCD23 profile and suggests that human beings may be classified into high and low IgE responders on the basis of IL-4 production.

Adolescent↗

PubMed1993

[Food allergy: identifying and characterizing peanut allergens with patient sera and monoclonal antibodies].

The purpose of this study is to improve the diagnosis of peanut allergy. In order to standardize test substances for in vivo and in vitro diagnostic, the type I allergy-associated single components of peanuts have been identified and characterized with the aid of patients' sera and monoclonal antibodies. For allergen detection IEF-immunoprint-, SDS-PAGE-immunoblot- and 2-D electrophoresis-techniques have been used. A comparison of control sera and patients' sera showed that both contained peanut specific IgG-, IgA- and IgM-antibodies. In contrast, peanut-specific IgE-antibodies were only detectable with patients' sera. In IEF-immunoprint the most intensive IgE-bindings showed up in pl-range from pH 5.5 to 7.5. In SDS-PAGE-immunoblot major allergens could be identified at molecular weight ranges of 17, 30, 48 to 66 and 116 kD. Raising monoclonal antibodies against IgE-reactive components from peanut extract resulted in eight antibody-producing hybridoma cell lines, named PN-a to PN-h. ELISA-inhibition tests revealed common epitopes of monoclonal antibodies and patients' antibodies. Moreover, the monoclonal antibodies were tested to see whether they can be used for detection of hidden peanut allergens.

Allergens↗

PubMed1993

Characterization of grass group I allergens in timothy grass pollen.

Using Phl p V-depleted timothy grass pollen extract (Phleum pratense) as immunogen, we obtained a monoclonal antibody, QG 4, which recognized proteins of 33, 35, and 37 kd as determined by Western blotting. The antibody cross-reacted with pollen proteins of other grass species in the molecular weight range of 30 to 37 kd. By means of two-dimensional polyacrylamide gel electrophoresis blot of timothy grass pollen extract, we demonstrated at least seven protein spots: two of 37 kd with isoelectric points of 6.4 and 6.6; four of 35 kd with isoelectric points of 6.5, 6.8, 7.1, and 7.3; and one of 33 kd with an isoelectric point of 8.5. These protein spots were also detected by patients' pooled serum. Microsequencing of the 20 N-terminal amino acid residues revealed structures with sequence identities up to 90% to the well-established allergen, Lol p I of ryegrass (Lolium perenne). Therefore we assume that the monoclonal antibody QG 4 recognized the corresponding allergen Phl p I in timothy grass pollen.

Allergens↗

PubMed1993

Successful technique for the selective production of monoclonal antibodies against a major allergenic component in timothy pollen extract.

Monoclonal antibodies (MAbs) were selectively raised against a major allergenic component of 38 kD in timothy grass pollen (Phleum pratense). We used a special prefractionating technique to isolate the 38 kD allergen, because immunizations with crude pollen extract had resulted in a wide variety of antibodies against other determinants, and cross-reactions between different-sized proteins occurred. Pollen extract was separated by Western blotting. Strips of the nitrocellulose membrane containing the allergen were excised, dispersed into allergen bearing particles, and used for immunization of BALB/c mice. Five MAbs were obtained that reacted with the 38 kD allergen. Four of these antibodies (BF 1, DC 9, EB 4, GE 2) exclusively detected the 38 kD protein, while one MAb (EB 6) additionally bound to a 32 kD allergen. Epitope mapping with the 3 IgG 1 antibodies (BF 1, EB 6, DC 9) was performed by ELISA inhibition tests using the purified 38 kD component. The antibodies did not interfere with each other. This confirms that they bind to different sites of the 38 kD molecule. Only a weak inhibitory effect of the MAbs on the binding of patient's IgE was determined, we suppose that the MAbs do not bind to IgE reactive epitopes. For standardization and further characterization of the 38 kD protein by peptide mapping the MAbs will be useful tools.

Allergens↗

PubMed1993

Effects of light fluence and wavelength on expression of the gene encoding cucumber hydroxypyruvate reductase.

We have investigated the regulation of cucumber (Cucumis sativus) hydroxypyruvate reductase mRNA abundance in response to white-, red-, and far-red-light treatments. Following irradiation of dark-adapted cucumber seedlings with 15 min to 4 h of either white or red light and return to darkness, the mRNA level for the gene encoding hydroxypyruvate reductase (Hpr) in cotyledons peaks in the darkness 16 to 20 h later. The response of the Hpr mRNA level to total fluence of white light depends more directly on irradiation time than on fluence rate. In addition to this time-dependent component, a phytochrome-dependent component is involved in Hpr regulation in dark-adapted green cotyledons as shown by red-light induction and partial far-red-light reversibility. Parallel measurements of mRNA levels for the ribulose bisphosphate carboxylase/oxygenase small subunit and for the chlorophyll a/b-binding protein show that Hpr is the most responsive to short (about 60 min) white- and red-light treatments and that each mRNA has a characteristic pattern of accumulation in dark-adapted cotyledons in response to light.

Alcohol Oxidoreductases↗

PubMed1993

Fucose alpha 1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals.

The reactivity of sera from honeybee venom allergic patients with the N-glycan of phospholipase A2 was investigated using neoglycoproteins with an enzyme-linked immunosorbent assay. Of 122 sera with appreciable levels of IgE antibodies directed against bee venom as measured by radioallergosorbent test, 34 sera exhibited significant amounts of glycan-reactive IgE. These sera cross-reacted with the N-glycan from the plant glycoprotein bromelain. The interaction of IgE with the N-glycan from phospholipase could be inhibited with glycopeptides from bromelain which shares the alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine with bee venom phospholipase. Since defucosylated bromelain glycopeptides or glycopeptides containing a Man3GlcNAc2 oligosaccharide were not recognized by most of these sera, we conclude that alpha 1,3-fucosylation of the innermost N-acetylglucosamine residue of N-glycoproteins forms an IgE-reactive determinant. This structural element is frequent in glycoproteins from plants, and it occurs also in insects. It is suspected to be one of the major causes of the broad allergenic cross-reactivity among various allergens from insects and plants.

Allergens↗

PubMed1993

Comparison of four grass pollen species concerning their allergens of grass group V by 2D immunoblotting and microsequencing.

The identification and characterization of allergenic components is a vital step towards improving diagnosis and therapy. Members of the grass family (Poaceae) reveal a high cross-reactivity among each other caused by the close phylogenetical relationship. In order to investigate the variability between allergenic components, we studied the allergen grass group V, one of the major allergens. Pollen extracts of 4 different tribes (timothy grass (Phleum pratense)--Agrostidae, perennial rye grass (Lolium perenne)--Festuceae, meadow velvet (Holcus lanatus)--Aveneae, and rye (Secale cereale)--Triticeae) of the Festucoideae subfamily were separated by 2D PAGE and investigated by immunoblotting using patients' poolserum and monoclonal antibodies (raised against group V allergens of timothy grass pollen). The antibodies identify different allergens in the four grass species. The components vary from 30-50 kDa and pI 4.8-7.0. The eight NH2-terminal amino acids were determined and indicated high similarities between the different components. These results cast doubt on the suitability of classifying allergens into groups based only on their molecular mass, isoelectric point and N-terminal sequence analysis. It suggests to classify allergens according to their IgE-reactive epitopes.

Allergens↗

PubMed1993

[Characterization of allergens of timothy grass (Phleum pratense)].

By means of 2-D electrophoresis and subsequent immunoblotting allergens of timothy grass pollen are separated as single components. Via N-terminal sequencing, it is possible to classify these components as isoallergens of defined groups according to the allergen nomenclature. We are able to identify the allergens as Phl p I, Phl p IV, Phl p V and Phl p VI.

Allergens↗