Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites.

BACKGROUND: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity. OBJECTIVES: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE. METHODS: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes. RESULTS: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2. CONCLUSION: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part II. Experimental Production of Equine Antivenom against the Redback Spider.

This is the first report on large-scale experimental production of an equine antivenom against the redback spider (Latrodectus hasseltii) lived in Japan. We captured 10,000 redback spiders in Japan and prepared the toxoids of crude venom extract, mixed the toxoids with a mineral oil adjuvant, and immunized healthy horses repeatedly over a period of several weeks. Thereafter, we separated the horse plasma, purified the γ-globulin fraction, and stocked it as a purified antivenom concentrate. Consequently, we manufactured approximately 6,500 vials of a single-dose freeze-dried test lot from a portion of the purified γ-globulin fraction, equivalent to the extract derived from 520 spiders. This test lot had an antitoxin titer comparable to that of a similar drug commercially available overseas (a liquid preparation), and the other quality met all quality reference specifications based on the Minimum Requirements for Biological Products and other guidelines relevant to existing antivenom drug products in Japan.

Molecular diagnosis and immunotherapy.

PURPOSE OF REVIEW: To describe recent insights into how molecular diagnosis can improve indication and selection of suitable allergens for specific immunotherapy and increase the safety of this therapy. RECENT FINDINGS: As specific allergen immunotherapy targets specific allergens, identification of the disease-eliciting allergen is a prerequisite for accurate prescription of treatment. In areas of complex sensitization to aeroallergens or in cases of hymenoptera venom allergy, the use of molecular diagnosis has demonstrated that it may lead to a change in indication and selection of allergens for immunotherapy in a large proportion of patients when compared with diagnosis based on skin prick testing and/or specific IgE determination with commercial extracts. These changes in immunotherapy prescription aided by molecular diagnosis have been demonstrated to be cost-effective in some scenarios. Certain patterns of sensitization to grass or olive pollen and bee allergens may identify patients with higher risk of adverse reaction during immunotherapy. SUMMARY: Molecular diagnosis, when used with other tools and patients' clinical records, can help clinicians better to select the most appropriate patients and allergens for specific immunotherapy and, in some cases, predict the risk of adverse reactions. The pattern of sensitization to allergens could potentially predict the efficacy of allergen immunotherapy provided that these immunotherapy products contain a sufficient amount of these allergens. Nevertheless, multiplex assay remains a third-level approach, not to be used as screening method in current practice.

The principle of homologous groups in regulatory affairs of allergen products--a proposal.

Among other legal regulations, the Note for Guidance on Allergen Products CPMP/BWP/243/96 released by the European Medicines Agency provides regulatory instructions regarding the quality of allergen extracts for diagnostic or therapeutic purposes. The current revision of this guideline intends to transform the so-called 'principle of taxonomic families' to the 'principle of homologous groups'. According to this concept, the data of one allergen extract demonstrating stability, efficacy and safety can, to a limited extent, be extrapolated to other allergen extracts belonging to the same homologous groups. The present work proposes the formation of homologous groups for pollen species and animal-derived materials on the basis of similar biochemical composition and homology/cross-reactivity of allergens or allergen sources. Some tree pollen species could be assigned to three different homologous groups, some weed pollen species to one homologous group and numerous grass pollen species to one homologous group on condition that data rely on single defined representative species. A homologous group for mites is limited to the Dermatophagoides species and the grouping of vertebrate-derived materials such as dander could be possible under restrictions. The criteria for the formation of the proposed homologous groups are illustrated in detail to provide an opportunity for extending existing homologous groups by further species in case of new insights in allergens and cross-reactivity of allergen sources. In this way, the concept of homologous groups could serve as a dynamic tool in the regulation of allergen products.

Duration of allergen immunotherapy in respiratory allergy: when is enough, enough?

OBJECTIVES: To investigate the duration of effective inhalant subcutaneous immunotherapy (SCIT) reported in the published literature and to determine if any specific biomarkers or clinical predictors exist that may identify patients who will remain in long-term remission after discontinuing treatment. DATA SOURCES: Articles were selected from a search of the PubMed database from 1976 to 2006 using the search terms immunotherapy and allergen immunotherapy in combination with venom, allergic rhinitis, asthma, mechanism, efficacy, and duration, as well as articles known to the authors and referenced in review articles. STUDY SELECTION: Articles were selected if evaluation of efficacy of the primary allergic disease treated after discontinuation of SCIT was stated as one of the objectives of the study. RESULTS: The rate of relapse after discontinuing SCIT ranges from 0% to 55% of patients in the studies reviewed in this article. The length of the specific allergen immunotherapy and allergen type (ie, perennial vs seasonal) may be variables that affect the duration of clinical remission after cessation of SCIT. One study found the duration of SCIT efficacy after discontinuation depended on duration of treatment and correlated with decrease in skin test reactivity. CONCLUSION: Until specific tests or clinical markers are identified that will clearly distinguish between patients who will relapse from those who will remain in long-term clinical remission after discontinuing effective allergen immunotherapy, the decision to continue or stop immunotherapy must be individualized.

Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects.

This review highlights some of the research advances in anaphylaxis; hypersensitivity reactions to foods, drugs, and insects; and allergic skin disease that were reported primarily in the Journal in 2005. Although studies documented deficiencies in community management of anaphylaxis, guidelines and National Institutes of Health summary reports provide direction toward improved research and education. At least 9% of young children "outgrow" a tree nut allergy. Advances in food allergy diagnosis include reports of probability of reactions to peanut at various peanut-specific IgE concentrations and skin test response size and the utility of evaluating IgE binding to specific epitopes. Future food allergy treatments might include selection of "less allergenic" fruit cultivars, genetic silencing of major allergens, and treatment of allergic patients with Chinese herbal remedies. Osteopontin might be a useful biomarker for success of venom immunotherapy. Progress in our understanding of the immunology of atopic dermatitis and autoimmune urticaria has also been made. These observations will likely contribute toward optimizing management of these common allergic disorders.

Safety considerations in assessing the role of immunotherapy in allergic disorders.

Specific immunotherapy (SIT) is accepted as an effective treatment of allergic diseases when high quality extracts are used. However, this form of treatment can cause untoward reactions among which systemic reactions are the most severe. Although life-threatening reactions are rare and deaths exceptionally reported, SIT should be prescribed by allergists to patients with well defined characteristics, and administered with care by (or under the close supervision of) physicians trained to deal rapidly with the reactions. Reactions with standardised extracts occur mostly during the dose increase phase but they can be prevented using adapted schedules and premedication. During maintenance injections or when vial batches are changed, standardised extracts of known shelf-life usually result in a low rate of systemic reactions. Patients with asthma are more prone to develop systemic reactions, and allergens should not be administered to patients with a forced expiratory volume in 1 second (FEV1) under 70% of predicted or in those who have unstable or symptomatic asthma. Systemic reactions may be observed with all allergens and allergenic preparations although it appears that high molecular weight extracts may be safer.

Antigen-induced histamine-release in diagnosis of allergy.

Antigen-induced histamine-release from basophils was evaluated in 12 patients with hypersensitivity to grass, 14 patients with hypersensitivity to grains and 14 patients with hypersensitivity to insect-venom using a test with very high sensitivity. - Histamine-release upon stimulation with antigen was found in all patients with grass-hypersensitivity, in 12 of 14 patients with grain-hypersensitivity and in 10 of 14 patients with insect-venom-hypersensitivity. Additionally performed determination of total-IgE proved less valuable, whereas specific-IgE and skin-test showed a higher validity in hypersensitivity to grains.

Allergic responses to airborne allergens and insect venoms.

Exposure to environmental allergens leads to human sensitization and disease by two different routes: inhalation (i.e., pollen allergy) and parenteral administration (i.e., insect sting anaphylaxis). In both, the pathogenesis of disease involves specific IgE antibodies and mediator release from mast cells and basophils. The relevant allergens have been characterized and found to be proteins with a molecular mass that ranges from 15,000 to 40,000 daltons. Appropriate diagnostic methods, skin testing, basophil histamine release and IgE antibody measurements (RAST), have been developed. Appropriate immunotherapy (immunization with the relevant allergens) leads to an increase in IgG (blocking) antibody. This therapy has proved to be useful in inhalational allergy and is potentially curative in parenterally induced anaphylaxis.