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Biomedical subjects

Heimo Breiteneder

Publications and source records attributed to Heimo Breiteneder.

At least 19 recordsLinked to original sources

Delayed tumor onset and reduced tumor growth progression after immunization with a Her-2/neu multi-peptide vaccine and IL-12 in c-neu transgenic mice.

Passive immunotherapy with monoclonal antibodies is a routinely performed but cost intensive treatment against certain cancers. Induction of humoral anti-tumor responses by active peptide immunization has therefore become a favorable treatment concept. We have recently identified three peptides representing B-cell epitopes of the extracellular domain of Her-2/neu each of them inducing Her-2/neu specific immune responses with anti-tumor activity in vitro. The present study was performed to evaluate the in vivo protective capacity of a combined vaccination with these three peptides in FVB/N transgenic mice spontaneously developing c-neu overexpressing breast cancers. The three Her-2/neu peptides coupled to tetanus toxoid were administered with or without addition of recombinant IL-12. At the time all untreated mice had developed tumors about 40% of peptide-immunized mice and nearly 60% of mice immunized with the peptide vaccine co-applied with IL-12 remained tumor free. Moreover, co-administration of IL-12 had a significant impact on the retardation of tumor progression. The enhanced anti-tumor efficacy of the vaccine by IL-12 was associated with a Th1 biased immune response as demonstrated by an increased IFN-gamma production in vitro and elevated Her-2-specific IgG levels. Our findings clearly demonstrate that this multi-peptide vaccine is effective in tumor prevention and support its use against minimal disease, drug-resistant tumors or even for prophylaxis against cancers overexpressing Her-2/neu.

Adjuvants, Immunologic↗

Nomenclature and structural biology of allergens.

Purified allergens are named using the systematic nomenclature of the Allergen Nomenclature Sub-Committee of the World Health Organization and International Union of Immunological Societies. The system uses abbreviated Linnean genus and species names and an Arabic number to indicate the chronology of allergen purification. Most major allergens from mites, animal dander, pollens, insects, and foods have been cloned, and more than 40 three-dimensional allergen structures are in the Protein Database. Allergens are derived from proteins with a variety of biologic functions, including proteases, ligand-binding proteins, structural proteins, pathogenesis-related proteins, lipid transfer proteins, profilins, and calcium-binding proteins. Biologic function, such as the proteolytic enzyme allergens of dust mites, might directly influence the development of IgE responses and might initiate inflammatory responses in the lung that are associated with asthma. Intrinsic structural or biologic properties might also influence the extent to which allergens persist in indoor and outdoor environments or retain their allergenicity in the digestive tract. Analyses of the protein family database suggest that the universe of allergens comprises more than 120 distinct protein families. Structural biology and proteomics define recombinant allergen targets for diagnostic and therapeutic purposes and identify motifs, patterns, and structures of immunologic significance.

Air Pollution, Indoor↗

Single-chain antibodies to the EWS NH(2) terminus structurally discriminate between intact and chimeric EWS in Ewing's sarcoma and interfere with the transcriptional activity of EWS in vivo.

The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.

Amino Acid Sequence↗

Effects of established allergen sensitization on immune and airway responses after secondary allergen sensitization.

BACKGROUND: Spreading of sensitization with clinical manifestation of allergy is often observed in atopic individuals. OBJECTIVE: To investigate the effects of an established primary allergen sensitization on immune responses and airway inflammation/reactivity on secondary allergen sensitization and airway challenges in a murine model. METHODS: Balb/c mice were primarily sensitized intraperitoneally with ovalbumin or PBS, followed by systemic sensitization and airway challenges with latex extract as a secondary, unrelated allergen. Purely sham-sensitized animals were included as controls. In a second set of experiments, the primary and secondary allergens were switched. RESULTS: Sensitization with ovalbumin before sensitization with latex resulted in increased production of total and latex-specific (Hev b 3-specific) IgE and IgG(1), and enhanced secretion of T(H)2-cytokines by spleen mononuclear cells cultured with mitogen compared with single latex-sensitized mice. Furthermore, airway challenges of double-sensitized mice (ovalbumin + latex) with latex caused a significant increase in airway reactivity compared with purely latex-sensitized and challenged animals. These effects were dependent on dosing and timing of the primary sensitization in relation to the secondary sensitization and independent of the primary allergen used. CONCLUSION: Primary sensitization boosted systemic T(H)2 immune responses and enhanced the development of airway reactivity after sensitization and airway challenges with a secondary, unrelated allergen. This effect of consecutive priming was dependent on the strength of the primary sensitization but independent of the allergen used. The results explain the increased susceptibility toward sensitization spreading in atopic individuals. CLINICAL IMPLICATIONS: Because sensitization spreading is facilitated by primary sensitization, early prevention measurements or immunotherapy should be considered at this stage of monosensitization.

Allergens↗

Apple allergy across Europe: how allergen sensitization profiles determine the clinical expression of allergies to plant foods.

BACKGROUND: Allergy to a plant food can either result from direct sensitization to that food or from primary sensitization to pollen, latex, or another food. OBJECTIVE: We sought to investigate the primary sensitizers in apple allergy across Europe, the individual allergens involved, and whether these differences determine the clinical presentation. METHODS: Patients (n = 389) with positive case histories and skin prick test responses to fresh apple were selected in the Netherlands, Austria, Italy, and Spain. Skin prick tests and RASTs to a panel of pollens and plant foods were performed, as well as RASTs to Bet v 1 and the apple allergens Mal d 1, 2, 3, and 4. RESULTS: In the Netherlands, Austria, and Italy apple allergy is mild (>90% isolated oral symptoms) and related to birch pollinosis and sensitization to Bet v 1 and its apple homologue, Mal d 1, which has an odds ratio of local reactions of 2.85 (95% CI, 1.47-5.55). In Spain apple allergy is severe (>35% systemic reactions) and related to peach allergy and sensitization to Mal d 3 (nonspecific lipid transfer protein), which has an odds ratio of systemic reactions of 7.76 (95% CI, 3.87-15.56). CONCLUSION: The analysis of individual apple allergens in a clinical context has provided insight into the sensitization pathway and into the intrinsic risk an allergen bears to induce mild or severe food allergy. CLINICAL IMPLICATIONS: Information on the sensitization pathway is essential to develop preventive strategies in food allergy. The application of individual food allergens with a known intrinsic risk will improve the prognostic value of diagnostic tests.

Adult↗

Structural bioinformatic approaches to understand cross-reactivity.

Cross-reactivity of allergens results from the presence of antibody-accessible conserved surface structures. These can best be studied when allergens have been structurally defined by X-ray crystallography or another structure determination method. When this is not the case, mimotope technology provides a useful alternative for elucidating antibody-binding sites on allergens. Structural bioinformatic approaches have been used to study the cross-reactivity of inhalant allergens with labile food allergens (Bet v 1 family) as well as the cross-reactivity between stable food allergens such as members of the nonspecific lipid transfer protein family. It was found that the degree of similarity of the structures correlated with the observed IgE cross-reactivities. However, IgE cross-reactivity between structurally unrelated allergens has not been demonstrated to date.

Allergens↗

Pollen allergens are restricted to few protein families and show distinct patterns of species distribution.

BACKGROUND: Inhalative allergies are elicited predominantly by pollen of various plant species. However, a classification of the large number of identified pollen allergens is still missing. OBJECTIVE: To analyze pollen allergen sequences with respect to protein family membership, taxonomic distribution of protein families, and interspecies variability. METHODS: Protein family memberships of all plant allergen sequences from the Allergome database were determined by using the Protein Families Database of Alignments and Hidden Markov Models. The taxonomic distribution of pollen allergens was established from the Integrated Taxonomic Information System. Members of abundant pollen allergen families were compared with allergenic and nonallergenic homologues by database similarity searches and multiple sequence alignments. RESULTS: Pollen allergens were classified into 29 of 7868 protein families. Expansins, profilins, and calcium-binding proteins constitute the major pollen allergen families, whereas most plant food allergens belong to the prolamin, cupin, or profilin families. Pollen allergens were revealed to be ubiquitous (eg, profilins), present in certain plant families (eg, pectate lyases), or limited to a single taxon (eg, thaumatin-like proteins). Allergenic plant profilins constitute a highly conserved family with sequence identities of 70% to 85% among each other but low identities of 30% to 40% with nonallergenic profilins from other eukaryotes, including human beings. Similarly, allergenic polcalcins possess sequence identities of 64% to 92% but show low identities of 39% to 42% to related nonallergenic calmodulins and calmodulin-like proteins from vegetative plant tissues and man. CONCLUSION: This classification of pollen allergens into protein families will aid in predicting cross-reactivity, designing comprehensive diagnostic devices, and assessing the allergenic potential of novel proteins.

Allergens↗

Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1.

BACKGROUND: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. METHODS: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. RESULTS: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. CONCLUSIONS: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

Allergens↗

Crystal structure of the major celery allergen Api g 1: molecular analysis of cross-reactivity.

Many patients who have been sensitised to pollen, display allergic symptoms after ingestion of certain plant food such as fresh fruit, vegetables and nuts. The cause is the cross-reactivity between structurally very similar major plant allergens. In particular, allergy to celery is very frequently associated with birch and mugwort pollen sensitization, known as to the birch-mugwort-celery syndrome. The crystal structure of the major celery allergen Api g 1, a homologue of the major birch pollen allergen Bet v 1, has been determined to a resolution of 2.9 A. The structure of Api g 1 is very similar to that of Bet v 1 with major differences occurring in the segment comprised of residues 23-45, preceding the well conserved glycine-rich P-loop, as well as in loops beta3-beta4 and beta5-beta6. In particular, Api g 1 lacks E45, which has been shown to be a crucial residue for antibody recognition in the crystal complex of Bet v 1 with the Fab fragment of a murine monoclonal IgG (BV16) antibody. The absence of E45 and the structural differences in the preceding segment suggest that this region of the Api g 1 surface is probably not responsible for the observed cross-reactivity with Bet v 1. A detailed analysis of the molecular surface in combination with sequence alignment revealed three conserved surface patches which may account for cross-reactivity with Bet v 1. Several residues of Bet v 1 which have been shown by mutagenesis studies to be involved in IgE recognition belong to these conserved surface regions. The structure of Api g 1 and the related epitope analysis provides a molecular basis for a better understanding of allergen cross-reactivity and may lead to the development of hypoallergens which would allow a safer immunotherapy.

Allergens↗

Specific IgE response to purified and recombinant allergens in latex allergy.

BACKGROUND: In recent years, allergy to natural rubber latex has emerged as a major allergy among certain occupational groups and patients with underlying diseases. The sensitization and development of latex allergy has been attributed to exposure to products containing residual latex proteins. Although improved manufacturing procedures resulted in a considerable reduction of new cases, the potential risk for some patient groups is still great. In addition the prevalent cross-reactivity of latex proteins with other food allergens poses a major concern. A number of purified allergens and a few commercial kits are currently available, but no concerted effort was undertaken to evaluate them. METHODS: We studied 11 purified latex allergens, Hev b 1 to Hev b 10, and Hev b 13 along with several crude allergen extracts and two commercial ImmunoCAP assays to evaluate specific IgE antibody in the sera from latex allergic patients and controls. Health care workers and spina bifida patients with clinical symptoms of latex allergy, spina bifida patients without latex allergy, and non-atopic health care workers have been studied. RESULTS: The results suggest that Hev b 2, 5, 6, and 13 together identified over 80 percent health care workers with latex allergy, while Hev b 6 along with Hev b 1 or 3 detected specific IgE antibody in all sera studied from patients with spina bifida and latex allergy. The ImmunoCAP results using both Hev b 5 amplified and non-amplified closely agreed with the clinical diagnosis of latex allergy in health care workers and in spina bifida. CONCLUSION: Although the purified allergens and crude extracts reacted diversely with IgE from different patient groups, the results indicated that use of certain combinations of purified recombinant antigens will be useful in commercial kits or in in-house assays for detecting specific IgE antibody in the sera. The results suggest that a combination of Hev b 2, 3, 5, 6, and 13 together detected specific IgE in 80% of the sera from latex allergic patients. Both ImmunoCAPs correctly identified over 95% of latex allergic patients, however, showed reactivity with a few normal control subjects.

Journal Article↗

Suppression of human melanoma tumor growth in SCID mice by a human high molecular weight-melanoma associated antigen (HMW-MAA) specific monoclonal antibody.

The lack of efficacy of available therapies for the treatment of malignant melanoma has emphasized the need to develop novel therapeutic strategies to prevent melanoma growth. We have tested whether the anti-HMW-MAA mAb 225.28S is able to inhibit human melanoma tumor growth in SCID mice because in vitro data suggested that this antigen plays a role in spreading, migration and invasion of melanoma cells. Tumors were established by subcutaneous injection of the human melanoma cell line 518A2 into SCID mice. When tumors reached a size of 5 mm, the mAb 225.28S was administered intravenously 4 times in 3 day intervals at 100 microg/injection. Within 14 days after the first administration of the mAb 225.28S, tumor growth was reduced by 52% as compared to control mice. Three hundred and seven genes of >20,000 genes contained on the GeneChip were changed in their expression level at least 2-fold after administration of the mAb 225.28S. The encoded proteins were mostly components or modifiers of the extracellular matrix, tumor suppressors, and melanogenesis associated proteins. Surprisingly, the administration of the control mAb that did not lead to a significant tumor growth inhibition in vivo resulted in the modulation of two-thirds of these genes. This is the first report of suppression of human melanoma tumor growth in SCID mice by the mAb 225.28S. Our results suggest that anti-HMW-MAA mAbs may represent useful reagents to apply passive immunotherapy to patients with malignant melanoma.

Animals↗

Entamoeba histolytica: analysis of the trophozoite proteome by two-dimensional polyacrylamide gel electrophoresis.

The Entamoeba histolytica genome project carried out at TIGR and the Sanger Institute has produced a near-complete set of deduced open reading frame sequences. These data provide strong support for the identification of signals from proteomic analyses such as two-dimensional electrophoresis by protein sequencing and/or mass spectrometric methods. To carry out an initial investigation of the E. histolytica proteome, appropriate sample preparation and silver staining protocols were adapted. After preparation of protein extracts from E. histolytica HM-1:IMSS trophozoites, solubilized proteins were separated in the first dimension in IPG (immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis. Of the more than 1500 protein spots visualized, several landmark spots were isolated from the gels and identified by either tryptic cleavage and subsequent MALDI-TOF mass spectrometry or by protein sequencing.

Animals↗

Vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro.

Peptide mimics of a conformational epitope that is recognized by a mAb with antitumor activity are promising candidates for formulations of anticancer vaccines. These mimotope vaccines are able to induce a polyclonal Ab response focused to the determinant of the mAb. Such attempts at cancer immunotherapy are of special interest for malignant melanoma that is highly resistant to chemotherapy and radiotherapy. In this study, we describe for the first time the design and immunogenicity of a vaccine containing a mimotope of the human high m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then generated by coupling the most suitable candidate mimotope to tetanus toxoid as an immunogenic carrier. Immunization of rabbits with this vaccine induced a specific humoral immune response directed toward the epitope recognized by the mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26% lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate a possible application of this mimotope vaccine as a novel immunotherapeutic agent for the treatment of malignant melanoma.

Amino Acid Sequence↗

Crystallization and preliminary structure determination of the plant food allergen Pru av 2.

Thaumatin-like proteins (TLPs) have mostly been investigated in the context of their function as pathogenesis-related proteins and only in recent years have some of them been classified as allergens. Here, the purification and crystallization of the first allergenic TLP, Pru av 2, a 23.3 kDa protein isolated from ripe cherries, is reported. The crystals diffracted to 2.1 A resolution at a rotating-anode generator and were found to belong to space group P2(1), with unit-cell parameters a = 44.48, b = 41.04, c = 59.16 A, beta = 106.61 degrees and one molecule per asymmetric unit. In order to obtain high-resolution data, an annealing protocol was applied that improved the resolution limit from 1.6 to 1.3 A at a synchrotron.

Allergens↗

Plant food allergens--structural and functional aspects of allergenicity.

The three dominating plant food allergen groups belong to the prolamin and cupin superfamilies and to the family 10 of pathogenesis-related proteins. The prolamin superfamily comprises allergenic 2S albumins, nonspecific lipid transfer proteins and cereal alpha-amylase/trypsin inhibitors. These allergens have related structures and are stable to thermal processing and proteolysis. The cupin superfamily comprises the allergenic 7S and 11S globulin storage proteins from peanuts, soybean and tree nuts which are heat stable and can form immunogenicity enhancing aggregates. The Bet v 1 family of allergens includes tree pollinosis-associated food allergens with low stability which induce the symptoms of the oral allergy syndrome.

Allergens↗

Molecular properties of food allergens.

Plant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators.

Allergens↗

Structural relatedness of plant food allergens with specific reference to cross-reactive allergens: an in silico analysis.

BACKGROUND: The body of sequence and structural information on allergens and the sequence analysis of whole plant genomes are facilitating the application of bioinformatic approaches to identifying and defining plant allergens. OBJECTIVE: An in silico approach was used to quantify the distribution of plant food allergen sequences across protein families and to develop and apply a novel means of assessing conserved surface features important for IgE cross-reactivity. METHODS: Plant food allergen sequences were classified into Pfam families on the basis of sequence homology. Contact surface areas of selected proteins were calculated with MOLMOL by using a 1.4-A probe, corrected by removing contributions from IgE inaccessible main chains and side chains forming the ligand binding sites. RESULTS: A set of 129 food allergen sequences were classified into only 20 of 3849 possible Pfam families, with 4 families accounting for more than 65% of food allergens. Structural bioinformatic analysis of conserved exterior main chains and amino acid side chains in cross-reactive homologues of Bet v 1 and nonspecific lipid transfer proteins showed higher levels of similarity than shown by simple sequence comparisons. Thus, 75% of the Mal d 1 surface is likely to bind anti-Bet v 1 antibodies, compared with a sequence identity of approximately 56%. CONCLUSION: Most plant food allergens belong to only 4 structural families, indicating that conserved structures and biological activities may play a role in determining or promoting allergenic properties. Structural bioinformatic analysis shows that conservation of 3-dimensional structure should be included in any assessment of potential IgE cross-reactivity in, for example, novel proteins.

Allergens↗

Cross-reactivity of mimotopes with a monoclonal antibody against the high molecular weight melanoma-associated antigen (HMW-MAA) does not predict cross-reactive immunogenicity.

The high molecular weight melanoma-associated antigen (HMW-MAA) is highly expressed in advanced primary and metastatic melanoma. An epitope of the core protein of HMW-MAA is recognized by the murine monoclonal antibody (mAb) 225.28S. In this study, we aimed to characterize peptides that antigenically mimicked this epitope and to determine their efficacy as components of an HMW-MAA-based anti-melanoma vaccine. Therefore, we screened a constrained 10 mer phage display peptide library against mAb 225.28S. Selected phage-displayed peptides were then tested for their specificity for the antibody's antigen-binding site. DNA sequences coding for specific peptide ligands were determined. Binding of mAb 225.28S to HMW-MAA was inhibited in a dose-dependent manner by phage-displayed peptides from 51 to 83% and by synthetic peptides from 38 to 87%. Subsequently, the immunogenicity of the five mimotopes with the highest inhibition capacity was examined in rabbits. Immunizations with synthetic mimotopes conjugated to tetanus toxoid resulted in peptide-specific antibodies, but none of the highly antigenic mimotopes induced HMW-MAA cross-reactive antibodies. This report describes an example of disparity between antigenicity and cross-reactive immunogenicity, complicating the selection of potential vaccine candidates.

Animals↗