Search

The causative treatment of type I allergies is a long pursued goal in immunology. To design safe and efficient vaccine preparations, the interaction of the allergen and the symptom-inducing IgE antibodies still needs to be better understood. In this article, we describe the use of the phage display technique in allergy. It yields epitope mimics, so-called mimotopes, which can be employed for both, investigation of allergen–IgE interactions and definition of safe novel vaccines. [Copyright &y& Elsevier]

Natural rubber latex proteins are a well-recognized cause of type-I allergic reactions that increasingly afflict health-care workers, housekeeping personnel, and other persons using latex gloves or latex products. More than a dozen individual latex allergens have been identified of which eight have received an international nomenclature designation. To study the biochemical and immunological properties in detail, it is desirable to clone and express each of these allergens. Proteins that are involved in rubber synthesis are most likely confined to latex whereas other enzymes, such as chitinases or glucanases, are also present in fruits and may account for the symptoms observed in the latex-fruit syndrome. The available data on the molecular characterization of latex allergens presented here illustrate the importance of this research in the production of better diagnostic tests and, perhaps, tools for immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: As seen with the proto-oncogene Her-2/neu, antibodies targeting different parts of a receptor can have opposing effects. Depending on epitope specificity, in this case, tumor growth can be inhibited – but also enhanced. Therefore, the definition of molecular binding sites is of increasing importance in modern medicine. We here introduce a novel approach for binding site localization, utilizing information obtained by the phage display technique. This is a high throughput screening method for identification of peptide mimics, so called mimotopes, of any binding structure of interest. All target molecules whose structure is available in the RCSB Protein Data Bank can be scanned for mimotope matches on their surface. In this study, we present the matching results of five mimotopes defined for the epitope recognized by trastuzumab (Herceptin®), a humanized monoclonal antibody inhibiting tumor growth, on Her-2/neu. The localization thus obtained corresponds to the known trastuzumab epitope. We therefore suggest the algorithm as a novel way of binding site definition, circumventing co-crystallization experiments. [Copyright &y& Elsevier]

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes. [ABSTRACT FROM AUTHOR]

Background: Nitration of proteins on tyrosine residues, which can occur due to polluted air under "summer smog" conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. Methodology/Principal Findings: BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y107) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. Conclusions/Significance: These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes. [ABSTRACT FROM AUTHOR]

Stilbenes comprise a group of polyphenolic compounds, which exert inhibitory effects on various malignancies. The aim of this study was to evaluate the antitumor effects of a previously unreported stilbene derivative—3,3′,4,4′,5,5′-hexahydroxystilbene, termed M8—on human melanoma cells. Cell-cycle analysis of the metastatic melanoma cell line M24met showed that M8 treatment induces G2/M arrest accompanied with a dose- and time-dependent upregulation of p21 and downregulation of CDK-2 and leads to apoptosis. M8 induces the expression of phosphorylated p53, proteins involved in the mismatch repair machinery (MSH6, MSH2, and MLH1) and a robust tail moment in a comet assay. In addition, M8 inhibited cell migration in Matrigel assays. Shotgun proteomics and western analysis showed the regulation among others of paxillin, integrin-linked protein kinase, p21- activated kinase, and ROCK-1 indicating that M8 inhibits mesenchymal and amoeboid cell migration. These in vitro data were confirmed in vivo in a metastatic human melanoma severe combined immunodeficient (SCID) mouse model. We showed that M8 significantly impairs tumor growth. M8 also interfered with the metastatic process, as M8 treatment prevented the metastatic spread of melanoma cells to distant lymph nodes in vivo. In summary, M8 exerts strong antitumor effects with the potential to become a new drug for the treatment of metastatic melanoma. [ABSTRACT FROM AUTHOR]

Background: IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcϵRI in human intestinal epithelium. Methodology/Principal Findings: FcϵRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FceRIa positive epithelial cells co-expressed FcϵRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcϵRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcϵRI and to bind IgE, whereas confluent cells were negative for c-chains. Conclusions/Significance: Our data provide the first evidence that the components of a functional FceRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FceRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested. [ABSTRACT FROM AUTHOR]

Abstract: Toxoplasma gondii is a ubiquitous intracellular parasite affecting most mammals including humans. In epidemiological studies, infection with T. gondii and allergy development have been postulated to be inversely related. Using a mouse model of birch pollen allergy we investigated whether infection with T. gondii influences allergic immune responses to birch pollen. BALB/c mice were infected with T. gondii oocysts either before or at the end of sensitisation with the major birch pollen allergen Bet v 1 and thereafter aerosol challenged with birch pollen extract. During the acute phase of infection, clinical signs correlated with increased levels of serum TNF-α, IL-6, IFN-γ and anti-Toxoplasma-IgM. In the chronic phase, Toxoplasma-specific serum IgG, brain tissue cysts and high IFN-γ production in spleen cell cultures were detected. Mice infected prior to allergic sensitisation produced significantly less allergen-specific IgE and IgG1, while IgG2a levels were markedly increased. IL-5 levels in spleen cell cultures and bronchoalveolar lavage fluid were significantly reduced, and airway inflammation was prevented in these mice. Notably, in mice infected at the end of the allergic sensitisation process, systemic and local immune responses to the allergen were markedly reduced. T. gondii infection was associated with up-regulation of Toll-like receptor 2 (TLR2), 4, 9 and 11, as well as T-bet (a differentiation factor for Th1 cells) mRNA expression in splenocytes; moreover, enhanced TGF-β, IL-10 and Foxp3 mRNA expression in these cells suggested that regulatory mechanisms were involved in suppression of the allergic immune response. Kinetic studies confirmed the induction of Foxp3+CD4+CD25+ regulatory T cells preferentially during the chronic phase of T. gondii infection. Our data demonstrate that T. gondii exhibits strong immunomodulating properties which lead to prevention of allergic immune responses and thereby support the hygiene hypothesis. [Copyright &y& Elsevier]

Abstract: House dust mite allergy occurs in 10–20% of the population. Improvement of the present immunotherapy requires detailed knowledge about the structure of the allergens. Mimotopes selected from phage peptide libraries imitate the conformational epitopes of a natural allergen. The aim of our study was to generate epitope mimics for the two major allergens of house dust mite. When the monoclonal anti-Der p 1 and anti-Der p 2 antibodies were used for biopannings, mimotopes were selected which bound also specific IgE from human allergic patients’ sera. The conformational matching of these mimotopes on the 3D structure of the natural allergens determined discontinuous epitopes in both cases, representing conformational B-cell epitopes relevant for binding of human IgE. Therefore, these mimotopes are potential candidates for the directed induction of blocking antibodies and epitope-specific immunotherapy of mite allergy. [Copyright &y& Elsevier]

Abstract: Based on two clinical observations of adverse reactions during exercise with latex sport bands, we aimed to assess the possible risk for allergic patients posed by this equipment by investigating allergen content and IgE binding potential. Protein extracts of three different latex sport bands were characterized with sera of latex allergic patients. The IgE recognition profile of the allergic patients was identified by component resolved diagnosis and the allergen composition of the extracts was characterized by inhibition assays with the recombinant latex allergens Hev b 1, 3, 5, 6.02, and 8. The sera showed pronounced IgE binding to all three blotted extracts, however with diverse patterns. Inhibition assays revealed the presence of Hev b 1, 3, 5, and 8 in latex sport band extracts. The clinical relevance of contained allergens was demonstrated by strong skin reactions when testing with latex sport bands. From our results we conclude that latex sport bands contain clinically relevant allergens and may cause latex allergic individuals to experience allergic symptoms, potentially amplified by exercise-induced mechanisms. Even though latex is labeled on products, it is important that patients as well as athletic trainers and physical therapists recognize the risk of adverse reactions with these bands. [Copyright &y& Elsevier]

Abstract: Induction of peripheral tolerance can be facilitated when the antigen is linked to the B subunit of cholera toxin (CTB), an efficient mucosal carrier. In the present study, a genetic fusion molecule of Bet v 1 and CTB was produced to test whether mucosal application of this construct would lead to suppression of Th2 responses. Intranasal pretreatment of BALB/c mice with rCTB-Bet v 1 prior to allergic sensitisation with the allergen significantly decreased IgE but markedly increased allergen-specific IgG2a levels in sera as well as IFN-γ production of splenocytes. This Th1 shift was supported by an increased T-bet/GATA3 mRNA ratio. IL-5 production within the airways was suppressed after the pretreatment with rCTB-Bet v 1, while local allergen-specific IgA antibodies were markedly enhanced by pretreatment with the construct. Upregulation of Foxp3, IL-10 and TGF-β mRNA expression was detected in splenocytes after pretreatment with unconjugated allergen but not with the fusion molecule, indicating that antigen conjugation to a mucosal carrier modifies the immunomodulating properties of an antigen/allergen. [Copyright &y& Elsevier]

The treatment of dyspeptic disorders with anti-acids leads to an increased risk of sensitization against food allergens. As these drugs are taken by 30-50% of pregnant women due to reflux and heart-burn, we aimed here to investigate the impact of maternal therapy with anti-acids on the immune response in the offspring in a murine model. Codfish extract as model allergen was fed with or without sucralfate, an anti-acid drug, to pregnant BALB/c mice during pregnancy and lactation. These mothers developed a codfish-specific allergic response shown as high IgG1 and IgE antibody levels and positive skin tests. In the next step we analyzed whether this maternal sensitization impacts a subsequent sensitization in the offspring. Indeed, in stimulated splenocytes of these offspring we found a relative Th2-dominance, because the Th1- and T-regulatory cytokines were significantly suppressed. Our data provide evidence that the anti-acid drug sucralfate supports sensitization against food in pregnant mice and favors a Th2-milieu in their offspring. From these results we propose that anti-acid treatment during pregnancy could be responsible for the increasing number of sensitizations against food allergens in young infants. [ABSTRACT FROM AUTHOR]

Abstract: Background: The role of anti-idiotypic antibodies in allergic disease is still poorly understood. According to Jerne, anti-idiotypic antibodies to IgE should represent internal images of an allergen. Our aim was to ultimately prove whether this hypothesis holds true in allergy. Here, we describe the selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual. Methods: Taking Phleum pratense grass pollen allergen Phl p 5 as a model, we selected anti-idiotypic antibodies against allergen-specific IgE directly from the B-cell repertoire of an allergic individual. We screened a combinatorial phage display library of human monovalent antibody heavy and light chain fragments (Fabs) with anti-Phl p 5a-IgE to identify and characterize Fabs with anti-idiotypic specificity. Results: Five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE were identified. Their hypervariable regions revealed partial sequence homology with solvent accessible antigenic sites of Phl p 5a, which have been identified by our previous mimotope approach. Phagemid DNA derived from the phage clones was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli. As a proof of molecular mimicry, both Fabs induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice. Molecular modeling of the heavy and light chain hypervariable loops of the anti-idiotypic Fabs illustrated structural similarity with dominant IgE epitopes of Phl p 5a. Conclusion: In this straightforward phage technology approach, antibodies with anti-idiotypic specificities could be isolated from a human allergic''s repertoire. As predicted by the immune network hypothesis, their hypervariable domains mimic IgE epitopes like internal images and, more importantly, induce allergen-specific immune responses in the absence of the allergen. [Copyright &y& Elsevier]

Abstract: Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients. [Copyright &y& Elsevier]

Background: The monoclonal antibody cetuximab (IMC-225, Erbitux) inhibits epidermal growth factor receptor (EGFR) signaling and has been approved for metastatic colon cancer therapy. However, to achieve effective titers, passive antibody therapies must be repeatedly administered over long periods. To overcome this limitation, we aimed to generate a vaccine inducing continuously available ‘cetuximab-like’ antibodies in vivo using the mimotope approach. Methods: We used the phage display technique to identify four peptides structurally mimicking the cetuximab epitope. We coupled two of these peptides to an immunogenic carrier protein, and we vaccinated four groups (n = 8) of BALB/c mice intraperitoneally with 10 μg of the mimotope conjugates, a control peptide conjugate, or the carrier protein alone. We assessed antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity mediated by the induced antibodies against EGFR-overexpressing human A431 carcinoma cells. We then tested receptor internalization capacity of the induced antibodies with fluorescently labeled EGFR, and we assayed their growth inhibitory potential toward A431 cells with a [³H]thymidine proliferation assay. Results: Mimotope-induced antibodies recognized EGFR, and both types of antibody-mediated cytotoxic effects were elicited by these antibodies. In both cellular cytotoxicity assays, the mimotope-induced antibodies exhibited specific lysis of more than 50%. The induced antibodies caused internalization of the receptor from the cell surface into endocytic vesicles and inhibited growth of EGFR-expressing cells to a similar extent as cetuximab [67% (95% confidence interval {CI} = 55% to 79%) and 69% (95% CI = 55% to 84%), respectively]. Conclusions: Epitope-specific immunization is feasible for active anti-EGFR immunotherapy. The in vitro biologic features of mimotope-induced antibodies are similar to those of the monoclonal antibody cetuximab. [ABSTRACT FROM AUTHOR]

Abstract: Neuraminidases act as a virulence factors for several pathogens that invade the human body through Peyer''s patch M-cells. Because of the structural similarity of Aleuria aurantia lectin (AAL) to neuraminidases, we hypothesized that AAL might also target human M-cells. In an in vitro human M-cell co-culture model significantly more particles were transported across the epithelium when microparticles were functionalized with AAL versus those that were not. Moreover, high concentrations of AAL induced no detectable cytotoxic effects on the related intestinal epithelial cell cultures, epithelial Caco2- and HT29-MTX-E12-cells. Upon incubation with AAL, PBMCs of allergic volunteers proliferated in response to AAL and secreted the cytokines, IL-2, IFN-γ, IL-10 and IL-5 in a concentration-dependent manner, indicating immune-stimulatory properties of the lectin. We conclude that AAL-coated microparticles may have the potential to target entrapped antigens to human M-cells for oral vaccination. [Copyright &y& Elsevier]

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Murine intestinal M-cells express α-l-fucose residues. We constructed α-l-fucose-targeting particles for oral immunotherapy of IgE-mediated allergy. Poly(d,l-lactic-co-glycolic acid)-microspheres were loaded with birch pollen allergens, and functionalised with the α-l-fucose specific Aleuria aurantia lectin (AAL). The AAL-microspheres had a size of 1–3μm, protected the entrapped allergens from gastric degradation and released 46.6±1.3% allergen over 21 days in vitro. Oral gavages of AAL-particles to naïve BALB/c mice induced birch pollen-specific IgG2a, but not IgG1 antibodies. We conclude that targeting allergens to α-l-fucose-receptor bearing cells using AAL-microspheres induces specific Th1-antibody responses possibly counteracting Th2-dominated allergy, and therefore provides a potentially useful formulation for oral immunotherapy. [Copyright &y& Elsevier]

Abstract: The disialoganglioside GalAcbeta1-4(NeuAcalpha2-8NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (GD2) is expressed on various tumors, including neuroblastoma, and was defined as a relevant tumor antigen. The monoclonal anti-GD2 antibody 14.18 is widely used for diagnostic purposes in neuroblastoma, and in its mouse/human chimeric form (ch14.18) now enters passive immunotherapeutic regimens in phase II clinical trials. This study aimed to generate structural mimics of the 14.18 epitope of GD2. Therefore, we used the ch14.18 antibody for selecting immunoreactive GD2 peptide mimotopes from a decamer phage display library. In all, 13 GD2 peptide mimics could be determined by biopanning and their specificity was demonstrated by exclusive recognition by the ch14.18 antibody. Furthermore, their nature of being GD2 mimics and their degree of mimicry was confirmed by competition with the natural antigen. When performing a comparative visualization of the GD2 epitope and selected mimotopes using a three-dimensional computer modeling system (BALLView), we demonstrated fitting of the GD2 molecule and the mimotopes in the antigen-binding pouch of a GD2 specific antibody. Moreover, the computer modeling argued for optimal affinity of the GD2 mimotopes. We thus provide evidence that the generation of GD2 peptide mimotopes is successful when using the neuroblastoma antibody ch14.18 for selection, and that this approach might offer a tool to develop a vaccination strategy against this malignant pediatric tumor. [Copyright &y& Elsevier]

Peptide mimotopes of tumor antigen epitopes have been proposed as components of tumor vaccines. In this study, we determined the immunogenicity of melcam mim1 and melcam mim2, peptide mimics of an epitope of the melanoma cell-adhesion molecule (Mel-CAM). BALB/c mice were vaccinated either with mimotopes or mimotopes coupled to tetanus toxoid (TT). The antibody responses of mice to melcam mim1, melcam mim2, and recombinant Mel-CAM were analyzed by an ELISA and immunoblot analyses. TT-coupled mimotopes led to high titers of IgG mainly of the IgG2a subclass to melcam mim1 and melcam mim2. Immunization with each of the mimotope formulations induced antibodies that cross-reacted with recombinant Mel-CAM. Uncoupled mimotopes induced lymphocyte proliferation and cytokine production in spleen cell cultures indicating that both peptide mimotopes also contained T cell epitopes. TT-coupled mimotopes induced T helper (Th)1 (interleukin (IL)-2, interferon-γ) and Th2 (IL-4, IL-5) cytokines, whereas uncoupled mimotopes induced a Th1-biased T cell response. Our results suggest that mimotopes potentially represent a novel vaccine approach to induce a tumor antigen-specific humoral and cellular response. [ABSTRACT FROM AUTHOR]

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coliin tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 μg rLTB pentamers per gram fresh plant material and was capable of binding GM1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 μg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity. [Copyright &y& Elsevier]

In this study, we constructed particles applicable for oral immunotherapy of type I allergy by protecting allergens from digestion and supporting intestinal antigen uptake. Therefore, birch-pollen allergens were entrapped in poly(d,l-lactic-co-glycolic acid) microspheres by spray-drying rendering microspheres with a main population of 1–3 μm. Microspheres were further coated with wheat germ agglutinin (WGA) to target enterocytes. Coating with WGA did not alter the surface characteristics of the microspheres as demonstrated in scanning electron microscopy. Binding of WGA was specific and could be inhibited by chitotriose to 14.7 ± 6.9%. Comparable amounts of allergen were released from both particle-types with 46.3 ± 1.7% and 44.5 ± 2.6% during 21 days. Simulating gastric digestion in vitro, antigenicity of allergens entrapped in WGA-microspheres was preserved to 59.8 ± 1.5% even after 2 h. Feedings of BALB/c mice with WGA-microspheres induced higher levels of allergen-specific IgG-levels than gavages of uncoated microparticles or naked protein. Thus, we conclude that WGA-microspheres are suitable vehicles for oral delivery and mucosal targeting due to lectin-mediated bioadhesion. [Copyright &y& Elsevier]

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses. [Copyright &y& Elsevier]

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA''s open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9 Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein''s eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N. benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens. [Copyright &y& Elsevier]

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9 A˚ using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development. [Copyright &y& Elsevier]

The cell surface adhesion molecule Mel-CAM is highly expressed in advanced primary and metastatic melanoma. Mel-CAM was first described as an integral membrane glycoprotein of malignant melanoma cells. The murine monoclonal antibody MAd18-5D7 recognizes an epitope of the extracellular domain of Mel-CAM and is able to enhance Mel-CAM mediated adhesion of melanoma cells in aggregation assays. For the characterization of peptides that antigenically mimic surface-exposed areas of Mel-CAM we screened a newly constructed random pVIII-28aa bacteriophage peptide library against MAd18-5D7. After three panning rounds a population of phages binding to MAd18-5D7 was enriched. Peptides expressed on the surface of these phages were then tested for their specificity for the antibody's antigen binding site. DNA sequences coding for two specific peptide ligands were determined. One of the deduced amino acid sequences showed similarity to a portion of the sequence of the third immunoglobulin-like extracellular domain of Mel-CAM. Both peptides blocked the interaction of MAd18-5D7 with Mel-CAM present in a MelJuSo melanoma cell line lysate. Phage displayed as well as synthetic peptides inhibited in a dose-dependent manner the binding of MAd18-5D7 to recombinant Mel-CAM in enzyme-linked immunosorbent assay experiments. No such inhibition was observed using a panel of other anti-Mel-CAM antibodies. Our results clearly indicate that these 28mer peptides are structural equivalents of the MAd18-5D7 epitope of Mel-CAM and that they will be useful tools for further in vitro and in vivo studies of Mel-CAM mediated cell–cell interaction. [ABSTRACT FROM AUTHOR]

Background: Plant profilins are important pan-allergens. They are responsible for a significant percentage of pollen-related allergies. Limited information is available about their involvement in the latex-fruit syndrome and the cross-reactivities between latex and pollen. We aimed to clone and express the Hevea brasiliensis latex profilin to investigate its allergological significance and serological cross-reactivities to profilins from plant foods and pollens. Methods: A DNA complementary to messenger RNA (cDNA) coding for the Hevea latex profilin, Hev b 8, was amplified by polymerase chain reaction from latex RNA. Recombinant (r)Hev b 8 was produced in Escherichia coli and used to screen sera from 50 latex- allergic health care workers (HCWs) with well-documented histories of food and pollen allergy and 34 latex-allergic spina bifida (SB) patients. The cross-reactivity of natural Hev b 8 and rHev b 8 with other plant profilins was determined by ELISA inhibition assays. A three-dimensional homology model of Hev b 8 was constructed based on known profilin structures. Results: The cDNA of Hev b 8 encoded a protein of 131 amino acids with a predicted molecular mass of 14 kD. Twelve of the 50 HCWs and 2 of the 34 SB patients were sensitized to Hev b 8. All Hev b 8-sensitized patients showed allergic symptoms to pollen or plant foods. Cross-reactivities between profilins of latex, pollen and plant food were illustrated by their ability to inhibit IgE binding to rHev b 8. Homology modeling of Hev b 8 yielded a structure highly similar to Bet v 2, the birch pollen profilin, with the most distinct differences located at the N-terminus. Conclusions: We conclude that primary sensitization to latex profilin in the majority of cases takes place via pollen or food profilins. Additionally, pollinosis and food-allergic patients with profilin-specific IgE can be at risk of developing latex allergy.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms. Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus. Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex. The aim of this study was to clone and express MnSOD from H. brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A. fumigatus and human MnSODs. Methods: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR. The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag. Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs. IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity. Results: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E. coli. The allergen was designated Hev b 10. The recombinant protein was enzymatically active, indicating the correct folding of the protein. In immunoblots, latex- as well as A. fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10. Cross-reactivity to Asp f 6, the MnSOD from A. fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10. Conclusions: Hev b 10 is a new cross-reactive allergen of H. brasiliensis which belongs to the ‘latex-mold’ group of latex allergens. Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho- d-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis , was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4 )2 SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients’ IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials. [ABSTRACT FROM AUTHOR]

Background: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients. In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level. Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1. Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features. Methods: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced. The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots. Results: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues. The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101. Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody. In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments. Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a. Total inhibition was only achieved when using purified natural Bet v 1. Conclusions: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens. Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

IgE-mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years. We purified natural Hev b 7, a 43-kDa patatin-like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences. A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library. The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa. The deduced amino acid sequence had 39-42 % identity to patatins from Solanum tuberosum. The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S. tuberosum, esterase activity. Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex-sensitized allergic individuals. In contrast to patatins from S. tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N-terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated. These results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins. [ABSTRACT FROM AUTHOR]

Focuses on profilin as a central regulatory component of the cytoskeleton. Isolation of profilin from trophozoites of the pathogenic Entamoeba histolytica strain SFL-3; Amino acid sequence; Actin-binding protein; Circular DNA cloning.

Individuals suffering from immediate hypersensitivity (type-I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type-l allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch-celery syndrome, in the form of a non-fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis-related and stress-induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross-reactivity with Bet v 1 was proven by cross-inhibition experiments, which provides further support for the existence of file birch-celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen. [ABSTRACT FROM AUTHOR]

The Bet v 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belongs to a group of conserved intracellular defense-related proteins that have been termed ribonuclese-like' PR proteins, on the basis of the partial sequence homology observed between PRI, a Bet v 1-homologue from parsley and a recently characterized ginseng ribonuclease. However, this enzyme activity has not yet been demonstrated, not for any of the members of this family of PR proteins nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v I using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro but show no activity on single or double- stranded DNA. [ABSTRACT FROM AUTHOR]

Background: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. Methods: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients’ sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. Results: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. Conclusion: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains. [ABSTRACT FROM AUTHOR]

Background: The prevalence of allergy to fruits and vegetables increased with pollinosis over the last 10 years. So far, clusters of hypersensitivity have been established and corroborated by the molecular characterization of individual cross-reacting allergens. Several case studies demonstrated the existence of allergic reactions to fruits of the subfamily Prunoideae (apricots, cherries, plums and peaches). Here, we present the characterization of a major allergen in cherry. Methods: Characterization was performed using IgE immunoblotting and immunoblot inhibition, N-terminal sequencing, mass spectroscopy analysis and PCR-based cDNA cloning. Results: A 23-kD protein was identified as IgE-binding component. As all cherry-extract-reactive sera displayed IgE-binding to this band, it was designated a major allergen from Prunus avium (Pru a 2). Sequencing the corresponding cDNA identified Pru a 2 as a thaumatin-like protein belonging to the group 5 of pathogenesis-related proteins. Conclusions: A thaumatin-like protein in cherry has been identified as a major allergen (Pru a 2). Homologous proteins from the thaumatin family share sequence similarities and should therefore be checked for the capability to elicit an IgE-mediated allergic reaction. [ABSTRACT FROM AUTHOR]

Background: An association of Crohn’s disease (CD) with food allergy has been discussed, but the role of food-specific IgE has not been clarified yet. Since CD is combined with immune complex formation, we examined in the present study whether anti-IgE autoantibodies in such complexes might hinder the determination of specific IgE. Methods: In order to elucidate the role of food-specific IgE in CD, we tested sera from CD patients (n = 107), healthy controls (n = 65) and allergics subjects (n = 7) for their IgE binding to food antigens (yeast, corn, celeriac, wheat) by an immunodot assay. After determining levels of IgE/IgG anti-IgE immune complexes, we purified them from serum pools of patients with CD, allergic subjects and healthy controls by affinity absorption using a monoclonal anti-IgE antibody. These purified immune complexes were treated by low pH (pH = 4) in order to dissolve them and to increase the detectability of food-specific IgE by RAST and CAP assay. Results: In CD sera no food-specific IgE could be detected, but levels of immune complexes of IgE and IgG anti-IgE autoantibodies were statistically significantly increased compared to healthy controls. pH treatment of purified IgE/IgG anti-IgE immune complexes resulted in a significant increase in specific IgE to yeast, corn, wheat and celeriac detected by RAST, however, only in the serum sample purified from allergic subjects. After pH treatment of CD immune complexes, specific IgE levels remained still very low. Conclusion: Thus, even if IgE seems to represent an autoantigen in CD, it is unlike to specifically participate in the pathophysiology of the putative food adverse reactions. [ABSTRACT FROM AUTHOR]

Purified preparations of allergenic proteins from plants, and in particular from pollens, consist of multiple closely related isoforms. These isoforms are highly similar in their amino acid sequences, yet they display different properties with respect to antibody binding, in this study we report of differential potencies of cross-reacting tree pollen allergens and cloned isoforms of these allergens to activate allergen-specific T-lymphocyte clones (T-cell clones: TCC). Six TCC with specifity for Bet v 1, a representative tree pollen major allergen, were established from peripheral blood of five birch-pollen-allergic donors. All TCC displayed the helper-cell phenotype. Five TCC reacted with distinct epitopes present on natural (n) and on recombinant (r) Bet v 1. One TCC could not be stimulated with r Bet v 1, in spite of strong reactivity with purified natural Bet v 1. The TCC were tested in proliferation assays using purified n Bet v 1, n Cot a 1 (the homologous major allergen of hazel pollen), r Bet v 1, four recombinant isoforms of Cor a 1 and peptides representing corresponding T-cell stimulating regions (isoepitopes) on these proteins. The clones showed different patterns of reactivity in response to stimulation with the five recombinant molecules and the corresponding peptides. Certain exchanges of amino acids within stimulating peptides correlated with a lack of proliferation of the TCC tested. These findings are important with respect to the use of broadly cross-reactive recombinant allergens or allergen-derived peptides for immunotherapy of type I allergy. [ABSTRACT FROM AUTHOR]

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding m these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor aI, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3′-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3′-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor aI. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor aI isoforms revealed identifies of 96–99%. The sequence identities between the Cor aI isoforms and Bet vI, the major birch pollen allergen, were 71–73% (80.5–83% similarity). Comparing amino acid sequences of Cor aI isoforms with the published sequences of Aln gI, the major allergen from alder, and Car bI and isoforms, the major allergen from hornbeam, 75.5–76.7% identity (83.6–85% similarity) and 83.6–89.9% sequence identity (89.3–95% similarity), respectively, was found. The four Cor aI cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor aI. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet vI) mAb, BIP 1. Our results demonstrate that Cor aI isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies. [ABSTRACT FROM AUTHOR]

Determines whether or not extracts of spices have direct effects on the permeability of intestinal epithelia and the transepithelial electrical resistance. Information relating to Solanaceae spices; Description of methodology used in study conducted; Highlights of the study; Analysis of data collected.

Presents a study on the ability of anti-ulcer drugs to promote immunoglobulin (IgE) formation toward dietary antigens in adult patients. Therapy that resulted in an increase in food-specific IgE in 25 percent of all treated patients; Experiments used to predict the allergenicity of food antigens through their resistance to gastric digestion; Foods in which the most resistant proteins were found.

Discusses a study which aimed to purify and characterize a glycosylated high molecular weight (HMW) allergen from celery, the major elicitor of food allergy in Central Europe. Description of HMW; Correlation between birch, mugwort and celery allergy; Evidence which concerns immunoglobulin E directed to HMW allergens.

Background: The induction of nonanaphylactogenic ‘blocking’ IgG antibodies capable of inhibiting the IgE/allergen interaction represents a favorable therapeutic concept for type I allergy. However, IgG antibodies to allergens may block or enhance specific IgE binding, depending on the recognized epitope. Taking the major birch pollen allergen Bet v 1 as a model, we developed a strategy for the precise induction of IgG antibodies of a desired epitope specificity. Methods: Random phage display peptide libraries were applied to define peptide structures mimicking natural epitopes (mimotopes) of Bet v 1. Selections were performed with BIP 1, a murine monoclonal antibody known to enhance the IgE binding to Bet v 1, and with anti-Bet v 1 IgE purified from patients’ sera. The characterized Bet v 1 mimotopes were used to localize the corresponding epitope at the surface of Bet v 1 by a computer-aided mathematical approach based on the three-dimensional structure and the chemical character of the amino acids. The Bet v 1 mimotopes were further used to immunize BALB/c mice. The specificity of the induced antibodies was tested by immunoblotting and inhibition assays. Results: With the three-dimensional epitope search it became possible to localize a discontinuous IgE epitope on the surface of Bet v 1 in a substantial distance from the IgG epitope of the monoclonal antibody BIP 1. Moreover, we could demonstrate that phage displaying mimotopes are immunogenic vectors for the precise induction of epitope-specific IgG. Immunization with BIP 1 mimotopes induced IgG enhancing the IgE binding to Bet v 1, whereas immunization with IgE mimotopes resulted in IgG capable of blocking human IgE binding in vitro. Conclusion: Allergen mimotopes can be used for the induction of anti allergen IgG of desired specificity. We propose that mimotope immunotherapy based on IgE mimotopes generated by biopannings may represent a future concept for therapy of type I allergy.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The 43–kD latex allergen Hev b 7 was purified from the latex of Hevea brasiliensis and identified by N–terminal and internal peptide sequences as highly homologous to patatins. Patatins are storage proteins encoded by a multigene family found in plants such as potato and tomato. We have obtained a cDNA clone coding for a cytoplasmic form of Hev b 7. The recombinant protein was expressed in the methylotrophic yeast Pichia pastoris at 10 mg/l culture supernatant. Both natural Hev b 7 and rHev b 7 were recognized by IgE in 11% of the latex–allergic patients. rHev b 7 inhibited binding to its counterpart in natural rubber latex extracts. Purified rHev b 7 used at concentrations of 10 μg/ml in skin prick tests produced wheal–and–flare reactions of sizes equal to those produced by nHev b 7. Furthermore, we were able to show that rHev b 7 possessed esterase activity. A plant expression system for the production of larger quantities of recombinant latex allergens as an alternative to the preparation from H. brasiliensis sap is discussed. [ABSTRACT FROM AUTHOR]