Fusion proteins consisting of Bet v 1 and Phl p 5 form IgE-reactive aggregates with reduced allergenic activity.

The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. METHODS: A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. RESULTS: A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. CONCLUSION: Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients.

Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.

BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

BACKGROUND: Group 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens. OBJECTIVE: To determine the binding sites and to study allergic patients' IgE recognition of the group 5 allergen (Phl p 5) from timothy grass pollen using human monoclonal IgE antibodies that have been isolated from grass pollen allergic patients. METHODS: Using recombinant isoallergens, fragments, mutants and synthetic peptides of Phl p 5, as well as peptide-specific antibodies, the interaction of recombinant human monoclonal IgE and Phl p 5 was studied using direct binding and blocking assays. Cross-reactivity of monoclonal IgE with group 5 allergens in several grasses was studied and inhibition experiments with patients' polyclonal IgE were performed. RESULTS: Monoclonal human IgE showed extensive cross-reactivity with group 5 allergens in several grasses. Despite its small size of 29 kDa, four independent epitope clusters on isoallergen Phl p 5.0101, two in each domain, were recognized by human IgE. Isoallergen Phl p 5.0201 carried two of these epitopes. Inhibition studies with allergic patients' polyclonal IgE suggest the presence of additional IgE epitopes on Phl p 5. CONCLUSIONS & CLINICAL RELEVANCE: Our results reveal the presence of a large number of independent IgE epitopes on the Phl p 5 allergen explaining the high allergenic activity of this protein and its ability to induce severe allergic symptoms. High-density IgE recognition may be a general feature of many potent allergens and form a basis for the development of improved diagnostic and therapeutic procedures in allergic disease.

High-resolution crystal structure and IgE recognition of the major grass pollen allergen Phl p 3.

BACKGROUND: Group 2 and 3 grass pollen allergens are major allergens with high allergenic activity and exhibit structural similarity with the C-terminal portion of major group 1 allergens. In this study, we aimed to determine the crystal structure of timothy grass pollen allergen, Phl p 3, and to study its IgE recognition and cross-reactivity with group 2 and group 1 allergens. METHODS: The three-dimensional structure of Phl p 3 was solved by X-ray crystallography and compared with the structures of group 1 and 2 grass pollen allergens. Cross-reactivity was studied using a human monoclonal antibody which inhibits allergic patients' IgE binding and by IgE inhibition experiments with patients' sera. Conformational Phl p 3 IgE epitopes were predicted with the algorithm SPADE, and Phl p 3 variants containing single point mutations in the predicted IgE binding sites were produced to analyze allergic patients' IgE binding. RESULTS: Phl p 3 is a globular ß-sandwich protein showing structural similarity to Phl p 2 and the Phl p 1-C-terminal domain. Phl p 3 showed IgE cross-reactivity with group 2 allergens but not with group 1 allergens. SPADE identified two conformational IgE epitope-containing areas, of which one overlaps with the epitope defined by the monoclonal antibody. The mutation of arginine 68 to alanine completely abolished binding of the blocking antibody. This mutation and a mutation of D13 in the predicted second IgE epitope area also reduced allergic patients' IgE binding. CONCLUSION: Group 3 and group 2 grass pollen allergens are cross-reactive allergens containing conformational IgE epitopes. They lack relevant IgE cross-reactivity with group 1 allergens and therefore need to be included in diagnostic tests and allergen-specific treatments in addition to group 1 allergens.

Possible therapeutic potential of a recombinant group 2 grass pollen allergen-specific antibody fragment.

The induction of blocking IgG antibodies that compete with IgE for allergen binding is one important mechanism of allergen-specific immunotherapy. The application of blocking antibodies may be an alternative treatment strategy. A synthetic gene coding for a single-chain fragment (ScFv) specific for the major timothy grass pollen allergen Phl p 2 was inserted into plasmid pCANTAB 5 E, and the recombinant ScFv was expressed in Escherichia coli and purified by affinity chromatography. The ScFv was tested for allergen binding by ELISA, and its association and dissociation were measured by surface plasmon resonance (Biacore) technology. The ability of the ScFv to inhibit allergic patients' IgE binding to Phl p 2 and Phl p 2-induced basophil degranulation was studied by ELISA competition and basophil activation (CD203c) assays. We report the expression, purification, biochemical and immunological characterization of a monomeric single-chain fragment (ScFv) of human origin specific for the major timothy grass pollen allergen, Phl p 2. The Phl p 2-ScFv showed high affinity binding to the allergen and blocked the binding of allergic patients' polyclonal IgE to Phl p 2 up to 98%. Furthermore, it inhibited allergen-induced basophil activation. The Phl p 2-ScFv inhibited allergic patients' IgE binding to Phl p 2 as well as Phl p 2-induced basophil activation and might be useful for passive immunotherapy of grass pollen allergy.

Recombinant allergen-based monitoring of antibody responses during injection grass pollen immunotherapy and after 5 years of discontinuation.

BACKGROUND: Subcutaneous injection immunotherapy (SCIT) is considered as antigen-specific and disease-modifying treatment with long-lasting effect. METHODS: We used a panel of recombinant grass pollen allergens for analyzing allergen-specific IgE, IgG(1) -IgG(4) , IgM, IgA, and light-chain (kappa, lambda) responses in grass pollen-allergic patients who had received one course of injection immunotherapy (SCIT) with an aluminum hydroxide-adsorbed grass pollen extract or only anti-inflammatory treatment. Serum samples were analyzed before and after 5 months of treatment as well as after 5 years. RESULTS: After 5 months of SCIT but not of anti-inflammatory treatment, IgG(1) > IgG(4) > IgG(2) > IgA antibody responses using both kappa and lambda light chains specific for major grass pollen allergens (Phl p 1, Phl p 5, Phl p 6, Phl p 2) increased significantly, whereas specific IgM or IgG(3) levels were unaltered. Allergen-dependent basophil degranulation was only inhibited with SCIT sera containing therapy-induced allergen-specific IgG antibodies. Likewise, decreases in Phl p 1- and Phl p 5-specific IgE levels and significant (P<0.001) reduction in symptom and medication scores were found only in the SCIT group but not in the group of patients receiving anti-inflammatory treatment. After 5 years, allergen-specific IgG antibody levels in the SCIT group had returned to baseline levels and there was no significant difference regarding symptoms between the SCIT and non-SCIT groups. CONCLUSION: The results from our observational study demonstrate that only SCIT but not anti-inflammatory treatment induces allergen-specific IgG and reduces boosts of allergen-specific IgE production but that one SCIT course was not sufficient to achieve long-term immunological and clinical effects.

Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5.

BACKGROUND: Allergen recognition by IgE antibodies is a key event in allergic inflammation. OBJECTIVE: To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5. METHODS: In a first step, the DNA sequence coding for the IgG(1) heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG(1) heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5. RESULTS: We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5. CONCLUSION AND CLINICAL RELEVANCE: Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.

Analysis of the antibody responses induced by subcutaneous injection immunotherapy with birch and Fagales pollen extracts adsorbed onto aluminum hydroxide.

BACKGROUND: Allergen-specific subcutaneous immunotherapy (SCIT) is an antigen-specific therapy of IgE-mediated allergies. In the present study, we analyze the epitope specificities of antibody responses induced by SCIT with allergen extracts from pollen of trees belonging to the order Fagales (birch, alder, hazel) adsorbed onto aluminum hydroxide. METHODS: The IgE, IgG1-4 and IgA responses to defined recombinant allergens (birch pollen: Bet v 1; alder pollen: Aln g 1; hazel pollen: Cor a 1; apple: Mal d 1) as well as to Bet v 1-derived recombinant fragments and synthetic peptides were analyzed in sera from patients who had undergone SCIT for different periods of time. RESULTS: Long-term SCIT (>1 year; cumulative dose >1,000,000 SQ units) induced more pronounced IgG1, IgG2 and IgG4 responses to Bet v 1 and Bet v 1-related allergens according to the degree of sequence homology (Bet v 1>Aln g 1>Cor a 1>Mal d 1) than short-term SCIT (<1 year; cumulative dose <1,000,000 SQ units). In contrast to patients treated for <1 year, patients treated for >1 year mounted distinct IgG1, IgG2 and IgG4 responses against sequential Bet v 1 epitopes. No relevant allergen-specific IgA or IgG3 responses were induced by short- or long-term SCIT. Using a competitive ELISA assay, it could be shown that serum IgG from patients undergoing long-term SCIT inhibited IgE reactivity to Bet v 1 better than IgG from patients undergoing short-term SCIT. CONCLUSION: SCIT with allergen extracts adsorbed onto aluminum hydroxide induces IgG responses against new epitopes that block IgE binding and cross-react with structurally related allergens depending, among other factors, on duration of treatment and cumulative injected dose.

Heterogeneity of commercial timothy grass pollen extracts.

BACKGROUND: The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources. OBJECTIVE: To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity. METHODS: Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients. RESULTS: The allergen extracts showed broad variations in protein compositions and amounts (24.1-197.7 microg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32-384 ng/mL; Phl p 2: 1128-6530 ng/mL, Phl p 5: 40-793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients. CONCLUSIONS: Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.

The Greater Involvement of People Living with AIDS principle: theory versus practice in Ontario's HIV/AIDS community-based research sector.

Drawing on the Greater Involvement of People with HIV/AIDS (GIPA) principle, the HIV/AIDS movement began to "democratize" research in Canada in the mid-1990s. To date, there is little evidence about the success of the community-based research (CBR) movement in relation to the implementation of GIPA. We draw on findings from a larger study examining barriers and facilitating factors in relation to HIV-related CBR in Ontario, Canada. An online survey was completed by 39 senior managers in Ontario AIDS service organizations (ASOs). Twenty-five in-depth, semi-structured interviews were then conducted to further explore the survey findings. Survey respondents reported that, compared to researchers and frontline service providers, people living with HIV/AIDS (PLWHA) tended to be the least involved in all stages (input, process and outcome) of CBR projects. AIDS service organizations with a mandate that included serving rural and urban communities reported even lower levels of PLWHA involvement in CBR. Qualitative data reveal complex barriers that make meaningful PLWHA engagement in CBR difficult, including: HIV-related stigma; health-related challenges; "credentialism"; lack of capacity to engage in research; other issues taking priority; and mistrust of researchers. Facilitating factors included valuing lived experience; training and mentoring opportunities; financial compensation; trust building; and accommodating PLWHA's needs. While there is strong support for the GIPA principles in theory, practice lags far behind.

Who benefits from community-based participatory research? A case study of the Positive Youth Project

Community-based participatory research (CBPR) has evolved as a popular new paradigm in health research. This shift is exciting, yet there is still much to discover about how various stakeholders are affected. This article uses a critical social science perspective to explore who benefits from these changes through an analysis of a CBPR case study (The Positive Youth Project). Two major categories of beneficiaries emerged: the research itself and the partner-stakeholders. The benefits, however, were not gained without substantial human resource investment, nor were they necessarily equitably spread. Participation costs included heavy demands of time, an added burden of work, frustration with the process, missing other opportunities, risking loss of anonymity, and loss of control. Care needs to be taken to ensure that concrete benefits accrue for all project partners and costs are minimized. Another way of framing benefits is to look at the community capacities built to address future health and social issues. (AU)

Molecular characterization of a human immunoglobulin G4 antibody specific for the major birch pollen allergen, Bet v 1.

BACKGROUND: Allergen-specific IgG4 antibodies induced by specific immunotherapy are thought to represent a protective immune response. Objective Our aim was the molecular characterization of a human IgG4 antibody (BAB5) specific for the major birch pollen allergen Bet v 1 that was derived from an immunotherapy-treated patient. METHODS: The cDNA coding for BAB5 was obtained by reverse transcriptase-PCR from the BAB5-producing cell line, compared with the germ line sequences and was expressed as a soluble antibody fragment in Escherichia coli. The epitope specificity and cross-reactivity of BAB5 were investigated with recombinant and synthetic Bet v 1 fragments and Bet v 1 homologous allergens from pollen. The ability of BAB5 to block allergic patients IgE was determined by competition experiments and sandwich ELISA. RESULTS: BAB5 is an affinity-matured Bet v 1-specific IgG4 antibody that reacts exclusively with Bet v 1 but not with Bet v 1-related allergens. Unlike an earlier-described monoclonal IgG1-blocking antibody, BAB1, which had been isolated from the same patient, BAB5 did not block allergic patients' IgE reactivity to Bet v 1. CONCLUSION: Our study demonstrates that not all allergen-specific IgG antibodies inhibit IgE recognition of allergens and can contribute to the success of immunotherapy. The epitope specificity and affinity of IgG antibodies but not their isotype are decisive for their protective activity.

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1.

Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli. The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3' end. The modified Bip1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip 1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v1 and, like Bet v1 -specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v 1 by particle blotting, (2) forthe detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy.

The immunoglobulin E-allergen interaction: a target for therapy of type I allergic diseases.

The interaction of immunoglobulin E and otherwise harmless antigens (allergens) leads in sensitized individuals through effector cell activation to the immediate induction of a cascade of inflammatory reactions, the hallmark of type I allergy. Recently, the molecular and structural characterization of allergens, specific IgE antibodies and their epitopes has made rapid progress. Here we discuss active and passive strategies for therapy of type I allergy, which are based on interfering with the IgE-allergen interaction.

Recombinant allergen-specific antibody fragments: tools for diagnosis, prevention and therapy of type I allergy.

Type I allergy represents a hypersensitivity occurring in almost 20% of the population that is based on the recognition of innocuous airborn antigens (pollen, mite, mould and pet allergens) by specific immunoglobulin E. Allergic symptoms (e.g. allergic rhinitis, conjunctivitis, asthma) are caused by the release of biological mediators from effector-cells after allergen-induced crosslink of receptor-bound IgE. Here we discuss strategies to obtain recombinant allergen-specific antibody fragments (Fabs) from mouse and human cell lines as well as directly from allergic patients lymphocytes via the combinatorial library technology. It is suggested to use recombinant allergen-specific Fabs for the standardization of allergen extracts currently used for diagnosis and treatment, to determine allergen contents in allergen sources and the environment to allow preventive measures and to use allergen-specific Fabs as therapeutic tools to interfere with the allergen-IgE interaction. The latter appears possible because IgE represents the least abundant class of immunoglobulins and there is increasing evidence for a limited diversity among allergens and their B-cell epitopes. Moreover, allergic effector reactions are mostly confined to accessible target organs so that a local application of competing Fabs prior to allergen exposure might represent a feasible therapeutic approach.

Tyrosine kinase signaling pathways control the expression of retinoic acid receptor-alpha in SK-BR-3 breast cancer cells.

Breast carcinomas are frequently characterized by hyperactivated c-erbB receptor tyrosine kinase signaling. Combination of anti-proliferative retinoids with growth-inhibitory c-erbB-specific agents might induce therapeutic benefit. We demonstrate close interactions between the c-erbB and the retinoic acid receptor system in SK-BR-3 breast cancer cells. Epidermal growth factor and heregulin-beta1 activate c-erbB receptors and dose- and time-dependently up-regulate retinoic acid receptor-alpha (RAR-alpha) mRNA. Similar effects have been found for the growth-inhibitory c-erbB-2 receptor tyrosine kinase-activating antibody 4D5 and the tyrosine phosphatase inhibitor orthovanadate. In contrast, the tyrosine kinase-inhibitor herbimycin A reduces tyrosine-specific protein phosphorylation and down-regulates RAR-alpha. Our data demonstrate that the expression of RAR-alpha, which represents a key mediator of the anti-proliferative effects of retinoids in breast cancer cells, is regulated by modulators of tyrosine kinase signaling. The levels of RAR-beta and -gamma mRNAs, however, are not affected by such agents.

Cloning allergen-specific antibody fragments (Fabs); tools for allergen standardization and therapy of type I allergy.

Recombinant allergens have made it possible to dissect the mechanisms of allergen-antibody interactions at a molecular level. It becomes clear that monoclonal human IgG antibodies as well as animal derived antibodies can block the interaction of specific IgE antibodies as well as the allergen induced allergic effector reaction. Using PCR technology and combinatorial plasmid vectors, recombinant antibody fragments can be produced and it has even become possible to isolate allergen-specific IgE Fabs out of combinatorial IgE libraries constructed from allergic patients lymphocytes. Recombinant Fabs will represent useful tools to study the IgE-allergen interaction as well as for the standardization of allergen extracts and quantitative allergen measurements. Moreover, allergen-specific recombinant Fabs which block the allergen-IgE interaction have to be considered as tools for local therapy in effector organs of allergic patients.

Expression of Zm13, a pollen specific maize protein, in Escherichia coli reveals IgE-binding capacity and allergenic potential.

Plant proteins belong to the most frequent elicitors of type I allergic symptoms in industrialized countries. Several relevant plant allergens have been found to be either specifically expressed or highly upregulated in mature pollen. The cDNA coding for a pollen specific maize protein, Zm13, shows significant sequence homology with a number of pollen or anther specific proteins from monocot and dicot plants as well as with recently described allergens from olive and rye grass. To test whether the Zm13 protein might possess IgE-binding capacity, Zm13 was expressed in E. coli. The coding region of Zm13 was PCR amplified from a genomic clone and expressed as a glutathione-S-transferase fusion protein. The recombinant Zm13 fusion protein bound a Zm13 specific rabbit antiserum and reacted with serum IgE from grass pollen allergic patients indicating that Zm13 and homologous proteins represent a family of conserved plant allergens.