Search PubMed⌕ Search

Biomedical subjects

Rudolf Valenta

Publications and source records attributed to Rudolf Valenta.

At least 37 records · Page 2Linked to original sources
PubMed2005

A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination.

BACKGROUND: Allergy vaccines based on natural allergen extracts contain greatly varying amounts of individual allergens with different immunogenicity. OBJECTIVE: To develop a novel type of allergy vaccine for complex allergen sources that combines defined amounts of the major allergens in the form of single hybrid molecules. METHODS: A hybrid molecule was engineered by PCR-based mending and expression of the cDNAs coding for the 4 major grass pollen allergens and compared with its single components by circular dichroism analysis, T-cell proliferation, ELISA competition, and histamine release assays. Immune responses to the hybrid molecule were studied in BALB/c mice and rat basophil leukemia assays. RESULTS: The hybrid contained most of the B-cell epitopes of grass pollen and could be used to diagnose allergy in 98% (n = 652) of patients allergic to grass pollen. Immunization of mice and rabbits with the hybrid induced stronger and earlier IgG antibody responses than equimolar mixtures of the components, which can be explained by the induction of stronger T-cell responses by the hybrid versus the individual components. IgG antibodies induced by vaccination with the hybrid blocked immediate allergic reactions, as demonstrated by rat basophil degranulation assays in a murine model of grass pollen allergy. CONCLUSION: We demonstrate for grass pollen allergy that recombinant hybrid molecules covering the spectrum of the disease-eliciting epitopes of complex allergen sources can be engineered.

Allergens↗
PubMed2005

IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation.

BACKGROUND: Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. OBJECTIVE: We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125-labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. RESULTS: Culture supernatants from activated allergen-specific T H 1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-gamma, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-alpha) identified IFN-gamma as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-gamma-mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. CONCLUSION: These results demonstrate that the T H 1 cell-derived cytokine IFN-gamma facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.

Allergens↗
PubMed2005

Intranasal tolerance induction with polypeptides derived from 3 noncross-reactive major aeroallergens prevents allergic polysensitization in mice.

BACKGROUND: Specific immunotherapy is less effective in patients with multiple allergic sensitizations compared with monosensitized patients. OBJECTIVE: We therefore established a mouse model of polysensitization to the major birch and timothy grass pollen allergens to test whether allergic polysensitization can be prevented by multiple allergen application via the mucosal route. METHODS: Female BALB/c mice were immunized intraperitoneally with recombinant (r) Bet v 1, rPhl p 1, and rPhl p 5. For intranasal tolerance induction, a mixture of the complete allergens was compared with allergen-derived immunodominant peptides applied either as a mixture or as a synthetic hybrid peptide composed of the T-cell epitopes of the 3 allergens. RESULTS: Intranasal application of the mixture of the complete allergen molecules did not prevent polysensitization to the same allergens. In contrast, pretreatment with a mixture of the immunodominant peptides or the hybrid peptide led to significantly reduced allergen-specific IgE responses in sera, IL-4 production in vitro, and suppressed airway inflammation. TGF-beta mRNA levels did not change, and IL-10 production was significantly suppressed after the pretreatment. The fact that the reduction of IL-10 was not abrogated after IL-10 receptor neutralization and that tolerance was not transferable with splenocytes indicates that the suppression of T(H)2 responses in polysensitized mice might not be mediated by immunosuppressive cytokines. CONCLUSION: Our study demonstrates that it is possible to suppress allergic immune responses simultaneously to several clinical important allergens. Thus, mucosal coapplication of selected peptides/hybrid peptides could be the basis of a mucosal polyvalent vaccine to prevent multiple sensitivities in atopic patients.

Allergens↗
PubMed2005

Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity.

BACKGROUND: We have performed a double-blind, placebo-controlled injection immunotherapy study with genetically modified derivatives of the major birch pollen allergen, Bet v 1 (Bet v 1-trimer, Bet v 1-fragments). OBJECTIVE: To investigate whether vaccination with genetically modified allergens induces allergen-specific antibodies in nasal secretions and to study whether these antibodies affect nasal allergen sensitivity. METHODS: A randomly picked subgroup of patients (n = 23; placebo, n = 10; trimer, n = 10; fragments, n = 3) was subjected to an extensive analysis of serum samples and nasal lavage fluids and to nasal provocation testing. Bet v 1-specific IgG(1-4) and IgA antibodies were determined in serum samples obtained before and after vaccination, after the birch pollen season, and 1 year after start of vaccination as well as in nasal lavage fluids obtained after the birch pollen season and 1 year after start of vaccination by ELISA. Nasal sensitivity to natural, birch pollen-derived Bet v 1 was determined by active anterior rhinomanometry after the birch pollen season and 1 year after start of vaccination. RESULTS: Vaccination with genetically modified Bet v 1 derivatives, but not with placebo, induced Bet v 1-specific IgG1, IgG2, and IgG4, and low IgA antibodies in serum, which also appeared in nasal secretions, but no IgG3 antibodies. The levels of therapy-induced Bet v 1-specific IgG4 antibodies in nasal secretions were significantly (P < .05) associated with reduced nasal sensitivity to natural, birch pollen-derived Bet v 1 as objectively determined by controlled nasal provocation experiments. CONCLUSION: Our data demonstrate that vaccination with genetically modified allergens induces IgG antibody responses against the corresponding natural allergen not only in serum but also in mucosal fluids, where they may protect against allergen-induced inflammation.

Adult↗
PubMed2005

Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1.

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.

Allergens↗
PubMed2004

Transition from a botanical to a molecular classification in tree pollen allergy: implications for diagnosis and therapy.

Tree pollens are among the most important allergen sources. Allergic cross-reactivity to pollens of trees from various plant orders has so far been classified according to botanical relationships. In this context, cross-reactivities to pollens of trees of the Fagales order (birch, alder, hazel, hornbeam, oak, chestnut), fruits and vegetables, between pollens of the Scrophulariales (olive, ash, plantain, privet, lilac) and pollens of the Coniferales (cedar, cypress, pine) are well established. The application of molecular biology methods for allergen characterization has revealed the molecular nature of many important tree pollen allergens. We review the spectrum of tree pollen allergens and propose a classification of tree pollen and related allergies based on major allergen molecules instead of botanical relationships among the allergenic sources. This molecular classification suggests the major birch pollen allergen, Bet v 1 as a marker for Fagales pollen and related plant food allergies, the major olive pollen allergen, Ole e 1, as a possible marker for Scrophulariales pollen allergy and the cedar allergens, Cry j 1 and Cry j 2, as potential markers for allergy to Coniferales pollens. We exemplify for Fagales pollen allergy and Bet v 1 that major marker allergens are diagnostic tools to determine the disease-eliciting allergen source. Information obtained by diagnostic testing with marker allergens will be important for the appropriate selection of patients for allergen-specific forms of therapy.

Allergens↗
PubMed2004

MAP kinase phosphorylation of plant profilin.

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.

Amino Acid Motifs↗
PubMed2004

Vaccine engineering improved by hybrid technology.

The term 'vaccination' describes the induction of protective immune responses against infectious diseases, but is also used to define antigen-specific forms of immunotherapy for allergy, cancer and autoimmunity. Successful vaccination requires either immune modulation or the induction of robust specific immunity to several disease-causing antigens. However, natural antigen sources may contain greatly varying amounts of these antigens and some of them may exhibit low immunogenicity. An approach for overcoming the latter problems has been developed for allergy vaccines recently. This approach is based on the genetic engineering of hybrid molecules, consisting of several major disease-eliciting antigens/epitopes. Such hybrid molecules can be built to include the most relevant epitopes of complex antigen sources. Moreover, fusion of different antigens in the form of hybrid molecules strongly increases their immunogenicity. The hybrid approach can also be used for the generation of mosaic antigens with altered immunological properties, which consist of re-shuffled antigen pieces. We exemplify the use of hybrid technology for the generation of new allergy vaccines and discuss its potential applicability for the development of vaccines for infectious diseases, cancer and autoimmunity.

Allergens↗
PubMed2004

Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.

The mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity are unknown. Single allergen-specific T cells constitute a very small fraction of the whole CD4+ T cell repertoire and can be isolated from the peripheral blood of humans according to their cytokine profile. Freshly purified interferon-gamma-, interleukin (IL)-4-, and IL-10-producing allergen-specific CD4+ T cells display characteristics of T helper cell (Th)1-, Th2-, and T regulatory (Tr)1-like cells, respectively. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4-secreting T cells in allergic individuals. Tr1 cells use multiple suppressive mechanisms, IL-10 and TGF-beta as secreted cytokines, and cytotoxic T lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all three allergen-specific subsets in different proportions, indicating that a change in the dominant subset may lead to allergy development or recovery. Accordingly, blocking the suppressor activity of Tr1 cells or increasing Th2 cell frequency enhances allergen-specific Th2 cell activation ex vivo. These results indicate that the balance between allergen-specific Tr1 cells and Th2 cells may be decisive in the development of allergy.

Adult↗
PubMed2004

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Allergens↗
PubMed2004

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7.

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

Allergens↗
PubMed2004

Biology of tree pollen allergens.

More than 25% of the population suffer from type I allergy. Pollens from trees of the Fagales, Oleaceae, and Cupressaceae belong to the most potent and frequent allergen sources. During the past 15 years, the nature of the most important allergens has been identified by molecular biological techniques, and recombinant allergens equivalent to the natural allergens have been produced. These advances provide insight into the biological functions of important allergens and allow the development of novel forms of diagnosis and therapy. In this review, we focus on Fagales allergens to illustrate the impact of recombinant allergens on diagnosis and therapy. We discuss structural similarities as a molecular basis for cross-reactivities and develop diagnostic concepts by using species-specific marker allergens as well as highly cross-reactive allergens. The identification of the allergen recognition profiles of patients with recombinant allergens allows a more precise selection of patients for available forms of allergy treatment. Moreover, we describe novel recombinant allergen-based forms of specific immunotherapy.

Allergens↗
PubMed2004

Genetically modified allergens.

For more than 90 years, allergen-specific immunotherapy, the only causative allergy treatment, has remained basically unchanged. The development of molecular biology techniques has led to the preparation of individual recombinant allergens from the most important allergen sources. Recombinant allergens can be used to determine the individual sensitization profile of allergic patients and have allowed the development of novel therapeutic tools. This article summarizes data on genetically modified recombinant allergen derivatives with reduced allergenic activity, which may be used to improve the safety and efficacy of specific immunotherapy.

Animals↗
PubMed2004

Human monoclonal antibody-based quantification of group 2 grass pollen allergens.

BACKGROUND: Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients. OBJECTIVE: The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens. METHODS: We have isolated a monoclonal human IgE Fab specific for group 2 grass pollen allergens by combinatorial cloning from lymphocytes of a grass pollen-allergic patient. This Fab was converted into a complete human IgG1 antibody and used together with rPh1 p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Ph1 p 2-specific IgG1 is incubated with a constant amount of biotinylated rPh1 p 2 competing with increasing concentrations of group 2 allergens to be determined. Defined concentrations of purified rPhl p 2 are used to establish a standard curve. The concentration of unlabeled group 2 allergens can thus be deduced from the displacement of biotinylated rPh1 p 2, which can be detected with peroxidase-labeled streptavidin. RESULTS: The competition-ELISA measured rPh1 p 2 concentrations ranging from 10 ng/mL to 500 ng/mL and allowed to quantify group 2 allergens from 9 different grass families. The results were in good agreement with immunoblot data. CONCLUSIONS: The described assay can be used for standardization of diagnostic and therapeutic vaccines as well as for the quantification of group 2 allergens in environmental samples.

Allergens↗
PubMed2004

FcepsilonRI engagement of Langerhans cell-like dendritic cells and inflammatory dendritic epidermal cell-like dendritic cells induces chemotactic signals and different T-cell phenotypes in vitro.

BACKGROUND: Atopic dermatitis (AD) is a biphasic inflammatory skin disease characterized by an initial phase predominated by T(H)2 cytokines, which switches into a second T(H)1-dominated chronic phase. Thus far, the small number of FcepsilonRI-bearing Langerhans cells (LCs) and inflammatory dendritic epidermal cells (IDECs) in the epidermis of patients with AD has hampered a detailed functional analysis and limited our knowledge of these dendritic cells (DCs). OBJECTIVE: We studied FcepsilonRI-mediated mechanisms of LCs and IDECs with the help of a novel in vitro model. METHODS: Langerhans cell-like dendritic cells (LC-DCs) and inflammatory dendritic epidermal cell-like dendritic cells (IDEC-DCs) bearing FcepsilonRI have been generated from monocytes of the same atopic donor and compared functionally with LCs and IDECs isolated from the skin of patients with AD. RESULTS: We found that FcepsilonRI-activated LC-DCs release chemotactic signals, and supernatants of FcepsilonRI-activated LC-DCs increase the migratory capacity of precursor cells of IDECs and naive T cells in vitro. FcepsilonRI-activated IDEC-DCs produce high amounts of proinflammatory cytokines and chemokines and might thereby amplify the inflammatory immune reaction in patients with AD. Furthermore, FcepsilonRI-activated IDEC-DCs prime naive T cells into IFN-gamma-producing T cells and release IL-12 and IL-18, which together might lead to the switch of the initial T(H)2-type immune response into a response of the T(H)1 type in vivo. CONCLUSION: The present study provides evidence that FcepsilonRI-activated LC-DCs and IDEC-DCs contribute distinctly to the outcome of T-cell responses in vitro and might have implications for the biphasic nature of AD in vivo.

Antigen Presentation↗
PubMed2004

Induction of autoallergy with an environmental allergen mimicking a self protein in a murine model of experimental allergic asthma.

BACKGROUND: Allergy and autoimmunity are traditionally considered as 2 exclusive entities related to the development of either TH2-dominated or TH1-dominated immune responses. OBJECTIVE: This study investigated whether allergic sensitization to a foreign antigen mimicking a self protein can induce allergy accompanied by an autoimmune response. METHODS: BALB/c mice were sensitized to human alpha-NAC, an evolutionary conserved component of the nascent polypeptide-associated complex, recently identified as an IgE-reactive autoantigen in patients with severe forms of atopy. By using nitrocellulose-blotted murine lung and skin extracts, purified recombinant human as well as murine alpha-NAC and murine alpha-NAC-derived synthetic peptides, the IgE, IgG1, and IgG2a antibody responses were measured, and their epitope specificity was mapped. RESULTS: Cross-reactivity of IgE and IgG antibodies with murine alpha-NAC was found in mice sensitized with human alpha-NAC. The biological relevance of the antibody response was demonstrated by the induction of immediate skin reactions in sensitized mice and by the fact that skin sensitivity could be passively transferred with serum to naive mice. Antigen challenge of sensitized mice resulted in airway inflammation accompanied by eosinophil and neutrophil accumulation, airway hyperresponsiveness to methacholine and perivasculitis of lung veins. CONCLUSION: Our data demonstrate that sensitization with a foreign antigen mimicking self can induce an allergic immune response of a mixed TH2 and TH1 profile that is associated with autoreactivity. Cross-sensitization to self may represent an important pathomechanism involved in the maintenance of severe and chronic forms of allergy.

Allergens↗
PubMed2004

Characterization of FcepsilonRI-bearing CD123 blood dendritic cell antigen-2 plasmacytoid dendritic cells in atopic dermatitis.

BACKGROUND: The high-affinity receptor for IgE (FcepsilonRI) on myeloid dendritic cells has been shown to play a major role in atopic dermatitis (AD). Plasmacytoid dendritic cells (pDCs), which are instrumental in the defense of viral infections, are present in reduced amounts in the skin of patients with AD, which is characterized by a high susceptibility to viral infections. OBJECTIVE: We explored phenotypical and functional characteristics of pDC in the peripheral blood of patients with AD and healthy individuals. METHODS: Blood dendritic cell antigen-2+CD123+ pDCs were enriched from the peripheral blood of patients with AD and studied in functional assays. RESULTS: Skin-homing molecules such as cutaneous lymphocyte antigen and L-selectin CD62L were expressed in lower levels on pDCs of patients with AD. pDCs expressed high amounts of IgE-occupied FcepsilonRI. Further, FcepsilonRI aggregation on pDCs impaired the surface expression of MHC I and II, induced the production of IL-10, and enhanced the apoptosis of pDCs. Importantly, FcepsilonRI preactivated pDC produced less IFN-alpha and IFN-beta after stimulation with CpG motifs and enhanced the outcome of immune responses of the TH2 type. CONCLUSION: From these data, we conclude that FcepsilonRI-bearing pDCs from patients with AD (1) are different from pDCs of healthy individuals, (2) might be important in the pathophysiology of AD, and (3) contribute to the enhanced susceptibility of patients with AD to viral infections.

Adult↗