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G Schramm

Publications and source records attributed to G Schramm.

At least 19 recordsLinked to original sources
PubMed2006

IPSE/alpha-1: a major immunogenic component secreted from Schistosoma mansoni eggs.

During infection with Schistosoma mansoni the egg stage of this parasite modulates the initial T helper (Th1) response into a Th2 response. This suggests that schistosome eggs contain factors responsible for that effect. We have recently described a glycoprotein (IPSE) from S. mansoni eggs that has a potent IL-4-inducing effect on human basophils. Here we demonstrate that IPSE is identical to a previously described molecule, the S. mansoni egg antigen alpha-1. We furthermore show that the expression of IPSE/alpha-1 at the level of both mRNA and protein is restricted to the egg stage. IPSE/alpha-1 is produced in and released from the subshell area of the egg and comes into close contact with inflammatory cells recruited to the vicinity of the egg surface. In line with this IPSE/alpha-1 is one of three major S. mansoni egg glycoproteins that induce pronounced antibody responses. Its IL-4-inducing capacity, moreover, suggests that IPSE/alpha-1 plays a role in initiating the Th2 response induced by patent S. mansoni infections.

Animals↗
PubMed2004

Echinococcus multilocularis metacestode extract triggers human basophils to release interleukin-4.

Infections with parasitic helminths are associated with a T helper 2 (Th2) immune response and IgE production. The underlying mechanism, however, is only partially understood. Recently we have isolated a protein from extracts of Schistosoma mansoni eggs that triggers human basophils from non-sensitized donors to release interleukin-4 (IL-4), the key cytokine of a Th2 response. We called this protein IPSE (for IL-4-inducing principle from Schistosoma mansoni eggs). Supposing that IPSE-like IL-4-inducing activities might be a general principle shared among different helminth species, we investigated extracts from the cestode E. multilocularis for its effect on human basophils. Our results showed that extracts from metacestodes of E. multilocularis cause basophil degranulation, as well as the secretion of histamine, IL-4 and IL-13, in a dose-dependent manner. IgE stripping and resensitization of basophils indicated that the mechanism of IL-4 induction requires the presence of IgE on the cells. Since analogous properties have been demonstrated earlier for IPSE, we think that S. mansoni and E. multilocularis may induce a Th2 response in their hosts via a related mechanism, namely, by the induction of IL-4 release from basophils.

Animals↗
PubMed2001

Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen.

The knowledge of IgE-binding epitopes on allergen molecules is important for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy. Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen. We performed an IgE-binding epitope mapping of rHol l 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragments (length between 15 and 186 amino acids), which were expressed in E. coli as MBP fusion proteins. Using sera of 65 grass pollen allergic patients, the fragments were analysed by immunoblotting for IgE reactivity. Specificity of antibody binding was confirmed by competitive blot inhibition assays. At least four different continuous IgE-binding epitopes were identified on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroyed by further fragmentation. The fragments were differentially recognized by individual patients' sera. By investigating IgE-binding to one of the small fragments in more detail, we found further epitope regions on this fragment. It was noteworthy that IgE reactivity to small fragments was weak compared to large fragments or to the complete molecule. Competitive blot inhibition experiments showed that binding of IgE antibodies to the small fragments was specific but with lower avidity than to the complete rHol l 5. rHol l 5 harbours multiple discontinuous as well as continuous IgE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients. Low avidity of IgE antibodies to small fragments suggests that the continuous epitope regions do not represent the complete epitope and are most probably parts of discontinuous epitopes.

Allergens↗
PubMed2001

A glycoprotein from Schistosoma mansoni eggs binds non-antigen-specific immunoglobulin E and releases interleukin-4 from human basophils.

We have recently shown that soluble extracts from Schistosoma mansoni eggs (SmEA) triggered basophils from nonsensitized donors to rapidly release interleukin (IL)-4. Assuming that this mechanism might play a role in vivo in biasing the immune response towards a Th2 phenotype, we determined basic properties of the IL-4-inducing activity contained in SmEA. Sensitivity to pepsin digestion indicated protein nature. Binding to and specific elution from Concanavalin A-sepharose suggested that this protein contains mannose residues, thus being a glycoprotein. The IL-4-inducing activity was stable for 30 min at room temperature towards shifting the pH between 3 and 10. When incubated at 100 degrees C, it was stable at pH 3, but less stable at neutral and alkaline pH. Electroelution from an SDS-PAGE gel indicated an apparent molecular weight of the IL-4-inducing activity between 31 and 66 kDa. Although binding to purified human immunoglobulin E (IgE) and activating basophils IgE-dependently, SmEA appears to activate basophils in a non-antigen-specific way, since the cells were purified from noninfected donors. Because the IL-4-inducing activity was found to be released from eggs, it could be an important factor in the environment of the eggs skewing the immune response towards the Th2 phenotype.

Animals↗
PubMed2001

Detection of mRNA for eotaxin-2 and eotaxin-3 in human dermal fibroblasts and their distinct activation profile on human eosinophils.

As many new biologically active chemokines have been cloned exploring the genomic DNA sequence database in the vicinity of already known chemokine sequences without demonstrating their natural origin, it is important to transfer findings from in vitro experiments with chemokines into the in vivo situation. With respect to eosinophils and fibroblasts that play an important part in the pathogenesis of allergic and autoimmune diseases, the role of the recently discovered members of the eotaxin family, eotaxin-2 and eotaxin-3, is not really understood. In order to elucidate the origin and biologic potency of the eotaxin family this study was performed. Conventional reverse transcription-polymerase chain reaction analysis was suitable to detect mRNA for eotaxin and eotaxin-3 but not for eotaxin-2 in dermal fibroblasts. In contrast to conventional reverse transcription-polymerase chain reaction, LightCycler analysis revealed that dermal fibroblasts constitutively expressed mRNA not only for eotaxin and eotaxin-3 but also for eotaxin-2. Moreover, with this technique we investigated mRNA expression levels after stimulation of fibroblasts with interleukin-4 and interleukin-4 plus tumor necrosis factor-alpha: the rank order of expression levels within the eotaxin family was eotaxin > eotaxin-3 > eotaxin-2. To address the question of the efficacy of eotaxin-3, we compared its activity with eotaxin, eotaxin-2, monocyte chemotactic protein-3, monocyte chemotactic protein-4, and RANTES in different test systems for eosinophils. The efficacy of the CC chemokines at equimolar concentrations with respect to the chemotactic response of human eosinophils was eotaxin-3 = eotaxin = eotaxin-2 > RANTES > monocyte chemotactic protein-4. The rank order of activity with respect to actin polymerization and release of toxic reactive oxygen species was eotaxin-3 = eotaxin = eotaxin-2 and eotaxin = eotaxin-2 > eotaxin-3 = monocyte chemotactic protein-3 = monocyte chemotactic protein-4 = RANTES, respectively. This study indicated a distinct profile in expression levels of the members of the eotaxin family in dermal fibroblasts. Indeed, all three eotaxin ligands demonstrated activation of human eosinophils with similar efficacies for chemotaxis, cytoskeletal rearrangements, activation of Gi proteins and transients of [Ca2+]i, but a distinct profile of activity with respect to the binding to CCR3 and the release of toxic reactive oxygen species. These findings may help to understand further the role of CC chemokines in fibroblast/eosinophil activation, which is of interest particularly in allergic and autoimmune diseases.

Actins↗
PubMed2000

A simple and reliable 5'-RACE approach.

A novel approach for the amplification of cDNA ends is described. It requires only minimal amounts of material, a simple cDNA synthesis reaction and a single PCR reaction to amplify the desired 5'- or 3'-ends of a certain cDNA of interest. It combines the so called CapFinder approach with solid phase cDNA synthesis, thus almost eliminating background problems usually associated with 5'-RACE protocols. This approach could be used to generate complete 5'-ends of numerous cDNAs using only one cDNA synthesis reaction. In combination with LA PCR, several kilobases of unknown 5'-ends could be amplified. It is easy to perform, quick, inexpensive and reliable, which should enable it to replace most currently used 5'-RACE protocols.

Animals↗
PubMed1999

"Allergen engineering": variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity.

One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.

Allergens↗
PubMed1999

Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils.

Dietary lectins, present in beans and other edible plant products, pose a potential threat to consumers due to their capacity to induce histamine release from basophils. In this study, we analyzed the capacity of 16 common, in particular dietary, lectins to induce human basophils to secrete IL-4 and IL-13, the key promoters of Th2 responses and IgE synthesis. Several of the lectins, especially concanavalin A, lentil lectin, phytohemagglutinin, Pisum sativum agglutinin and Sambucus nigra agglutinin, triggered basophils to release IL-4 at concentrations of up to 1 ng/10(6) basophils. Lectins with high IL-4-inducing capacity also stimulated the release of IL-13 and histamine. Lectin-induced IL-4 and IL-13 release reached a maximum after 4-6 h and more than 18 h, respectively. Affinoblotting revealed that lectins with the capacity to induce mediator release bind to IgE, suggesting IgE binding as initial step of signal generation. In conclusion, several dietary lectins can trigger human basophils to release IL-4 and IL-13. Since lectins can enter the circulation after oral uptake, they might play a role in inducing the so-called early IL-4 required to switch the immune response towards a Th2 response and type I allergy.

Basophils↗
PubMed1999

Early interleukin-4: its role in the switch towards a Th2 response and IgE-mediated allergy.

The etiology of IgE-mediated allergies is complex and, thus far, not completely understood. A common feature, however, is the overproduction of IgE-inducing cytokines, e.g. interleukin-4(IL-4), compared to IgE-antagonistic cytokines, such as interferon-gamma or IL-12. IgE-inducing cytokines are produced by T helper type 2 (Th2) cells. The differentiation of naive T cells towards the Th2 phenotype seems to be crucially dependent upon the particular cytokines present in the early stages of an immune response. Concerning the factors driving Th2 differentiation, the so-called 'early IL-4' seems to play an important role, although there is some controversy over the degree of its requirement and its cellular source. We have recently demonstrated that basophils might be such a source, since they rapidly release IL-4 upon antigen-specific or nonantigen-specific stimuli, such as certain lectins. This makes lectins interesting candidates for inducing a Th2 response and IgE-mediated allergy in unsensitized individuals.

Humans↗
PubMed1998

Molecular and immunological characterization of group V allergen isoforms from velvet grass pollen (Holcus lanatus).

Group V grass pollen allergens belong to the major grass pollen allergens causing reactions of type I allergy. cDNAs coding for two isoforms of the group V allergen of velvet grass pollen (Holcus lanatus), a widespread grass species, were isolated from a cDNA library by hybridization with a 5'-terminal reverse-transcribed PCR-derived cDNA probe. Amino acid sequences of the two isoforms, designated rHol l 5.01 and rHol l 5.02, revealed high similarity between them (78% identity) and to group V allergens and their isoforms found in other grass species. Recombinant Hol l 5 isoforms were expressed in Escherichia coli and purified as fusion proteins. To compare their immunological reactivities with group-V-specific monoclonal antibodies and patients' IgE, immunoblotting, ELISA and histamine release assay were performed. Interestingly, monoclonal antibody Bo9, specific for group Vb isoforms of timothy grass, bound only to isoform rHol l 5.01, not to rHol l 5.02. On the other side, IgE reactivities of patients' sera revealed no differences between the two isoforms when investigated by immunoblotting and only slight differences when investigated by ELISA. In histamine release assay both isoforms released comparable amounts of histamine from basophils of four individual patients. Thus, the two group V isoforms of velvet grass pollen exhibit differential binding when tested with monoclonal antibodies, i.e. different structure of single epitopes, but negligible differences concerning overall IgE-binding capacity and histamine-releasing capacity.

Allergens↗
PubMed1998

Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen.

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity; 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.

Allergens↗
PubMed1998

Allergenic activity of a major grass pollen allergen is elevated in the presence of nasal secretion.

Phl p5 is a major allergen of timothy grass and causes rhinitis and bronchial asthma in nearly all patients allergic to grass pollen. The biochemical processing of this molecule by the nasal mucosa at its first encounter and possible changes of its biologic activity are unknown. Two isoforms of the allergen were expressed in Escherichia coli and subsequently purified. Conversion of these preparations to various forms with molecular size between 10 and 20 kD in the presence of nasal secretion was observed. Surprisingly, in skin prick test assays with allergic patients the mixture of converted peptides caused significantly higher allergic response when compared with the parent protein. Allergenic activity of the recombinant N-terminal Phl p5a and the C-terminal Phl p5b as measured by skin prick test and histamine release assays was significantly higher than that of the respective parent molecules. Using pancreatic rather than nasal secretion, Phl p5b was completely degraded and its allergenicity was almost completely reduced. Proteolytic degradation converts Phl p5 to defined fragments with increased allergenicity. Complete degradation of Phl p5 on the mucosa could be a preventive strategy to destroy its potency for the induction of an allergic response.

Allergens↗
PubMed1997

Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity.

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups. Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.

Allergens↗
PubMed1997

A comparative randomized trial on the impact of two low-dose oral contraceptives on ovarian activity, cervical permeability, and endometrial receptivity.

In a double-blind randomized study, the suppression of ovarian activity and anti-conceptive effects on the cervix and endometrium were assessed during administration of two low-dose monophasic oral contraceptives (20 micrograms ethinyl estradiol [EE], 500 micrograms norethisterone--Eve 20 [Grünenthal, Aachen, Germany]; 20 micrograms EE, 150 micrograms desogestrel --Lovelle [Organon, Munich, Germany]). One hundred eighteen healthy women (ages: 18-35 years) were studied in 10 investigation centers during medication with either Eve 20 (n = 59) or Lovelle (n = 59). During three treatment cycles, ovarian activity was evaluated by sonographic determination of follicle-like structures (FLS) and by simultaneous assessment of serum endocrine profiles (gonadotropins LH and FSH, ovarian steroids estradiol [E2] and progesterone [P]). While on either treatment, no ovarian activity (as judged by no FLS and/or reduced sex steroid levels) was found in 90.8% (Eve 20) and 97.2% (Lovelle) of all investigated cycles. Follicular activity or cyst formation were detected in 18 of 173 cycles (Eve 20) and in 5 of 175 cycles (Lovelle), respectively. Gonadotropin levels were suppressed (LH < 6 IU/L, FSH < 8 IU/L) in most treatment cycles (Eve 20 76.6% vs. Lovelle: 84.8%). Serum E2 concentrations exceeding 0.1 nmol/L indicated residual follicular activity in 19.3% (Eve 20) versus 12.2% (Lovelle) of all cycles. An estimated by serum P levels over 5 nmol/L, ovulation had presumably occurred in 4.1% (Eve 20) versus 2.9% (Lovelle) of treatment cycles. However, when the sonographical and endocrinological data were combined, no ovulation was documented in any pill cycle. The quality and quantity of the cervical mucus was found to be minimal in the majority of women. Moreover, the endometrial layer was determined to be low by ultrasound during most pill cycles, indicating equally strong suppressive effects on endometrial receptivity by the two contraceptives. These observations suggest that ovarian activity is suppressed in the majority of cycles during use of low-dose contraceptives. This effect may mainly be medicated by pronounced suppression of serum gonadotropin levels. Strong anti-conceptive effects of these formulations on both cervical permeability and endometrial receptivity are additional factors ensuring the contraceptive efficacy of these formulations.

Adult↗
PubMed1997

Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol l 1.

BACKGROUND: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. OBJECTIVE: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1. METHODS: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients. RESULTS: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids). CONCLUSIONS: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

Allergens↗
PubMed1996

Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity.

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

Allergens↗
PubMed1996

Identification and characterization of the major allergens of velvet grass (Holcus lanatus), Hol l 1 and Hol l 5.

Although extract of velvet grass pollen is an important ingredient of commercial allergen extracts for immunotherapy, it is not defined concerning its major allergenic components. We have investigated the extract of velvet grass pollen by Western blotting using the sera of 97 patients and identified two major allergens of 34 and 30 kD. By two-dimensional immunoblotting with monoclonal antibodies and lectins, and by biochemical analysis the 34-kD protein was identified as a group I grass pollen allergen and the 30-kD protein as a group V grass pollen allergen. According to the nomenclature for grass pollen allergens we designated these allergens Hol l 1 and Hol l 5, respectively. Fragmentation of the velvet grass pollen extract by CNBr provided the first information on the localization of IgE binding epitopes on these two allergens. With Hol l 1 and Hol l 5 we identified two important components of commercial allergen extracts.

Allergens↗