Approaches to assess IgE mediated allergy risks (sensitization and cross-reactivity) from new or modified dietary proteins.

The development and introduction of new dietary protein sources has the potential to improve food supply sustainability. Understanding the potential allergenicity of these new or modified proteins is crucial to ensure protection of public health. Exposure to new proteins may result in de novo sensitization, with or without clinical allergy, or clinical reactions through cross-reactivity. In this paper we review the potential of current methodologies (in silico, in vitro degradation, in vitro IgE binding, animal models and clinical studies) to address these outcomes for risk assessment purposes for new proteins, and especially to identify and characterise the risk of sensitization for IgE mediated allergy from oral exposure. Existing tools and tests are capable of assessing potential crossreactivity. However, there are few possibilities to assess the hazard due to de novo sensitization. The only methods available are in vivo models, but many limitations exist to use them for assessing risk. We conclude that there is a need to understand which criteria adequately define allergenicity for risk assessment purposes, and from these criteria develop a more suitable battery of tests to distinguish between proteins of high and low allergenicity, which can then be applied to assess new proteins with unknown risks.

Precautionary allergen labelling: perspectives from key stakeholder groups.

Precautionary allergen labelling (PAL) was introduced by the food industry to help manage and communicate the possibility of reaction from the unintended presence of allergens in foods. However, in its current form, PAL is counterproductive for consumers with food allergies. This review aims to summarize the perspectives of all the key stakeholders (including clinicians, patients, food industry and regulators), with the aim of defining common health protection and risk minimization goals. The lack of agreed reference doses has resulted in inconsistent application of PAL by the food industry and in levels of contamination that prompt withdrawal action by enforcement officers. So there is a poor relationship between the presence or absence of PAL and actual reaction risk. This has led to a loss of trust in PAL, reducing the ability of consumers with food allergies to make informed choices. The result has been reduced avoidance, reduced quality of life and increased risk-taking by consumers who often ignore PAL. All contributing stakeholders agree that PAL must reflect actual risk. PAL should be transparent and consistent with rules underpinning decision-making process being communicated clearly to all stakeholders. The use of PAL should indicate the possible, unintended presence of an allergen in a consumed portion of a food product at or above any proposed action level. This will require combined work by all stakeholders to ensure everyone understands the approach and its limitations. Consumers with food allergy then need to be educated to undertake individualized risk assessments in relation to any PAL present.

Characterization of the Immunogenicity and Allergenicity of Two Cow's Milk Hydrolysates--A Study in Brown Norway Rats.

Hypoallergenic infant formulas based on hydrolysed milk proteins are used in the diet for cow's milk allergic infants. For a preclinical evaluation of the immunogenicity and allergenicity of new protein ingredients for such hypoallergenic infant formulas as well as for the investigation of which characteristics of hydrolysates that contribute to allergenicity, in vivo models are valuable tools. In this study, we examine the immunogenicity and allergenicity of two hydrolysates in a Brown Norway (BN) rat model, using i.p. dosing, which allows for the use of small quantities. Intact BLG, hydrolysed BLG and a hydrolysed whey product suitable for use in extensively hydrolysed formulas were thoroughly characterized for protein chemical features and administered to BN rats by i.p. immunization with or without adjuvant. Sera were analysed for specific IgG and IgE for evaluation of sensitizing capacity, immunogenicity and antibody-binding capacity. For evaluation of eliciting capacity a skin test was performed. The study showed that the hydrolysates had no residual allergenicity, lacking the capacity to sensitize and elicit reactions in the BN rats. Dosing with or without adjuvant induced a large difference in immunogenicity. Only antibodies from rats sensitized to intact BLG with adjuvant were able to bind the hydrolysates, and the whey-based hydrolysate only showed immunogenicity when dosed with adjuvant. This study showed that hydrolysates can be evaluated by an i.p. animal model, but that the choice of in vitro tests used for evaluation of antibody responses may greatly influence the result as well as may the use of adjuvant.

Induction of Bcl-xL-specific cytotoxic T lymphocytes in mice.

The induction of active immunity against tumour-associated antigens to prevent relapse of cancer is a promising approach but has so far shown only low efficacy. This low efficacy may in part be due to clonal escape of tumour cell variants by the downregulation of antigen expression or inflammation-induced dedifferentiation. Identification of novel tumour-associated antigens that at the same time are essential for continued tumour cell survival is thus critical for the development of active cancer vaccinations. At the same time, identification of novel endogenous murine tumour antigens will help improve preclinical development of cancer immunotherapy. The anti-apoptotic protein Bcl-xL has been suggested to be such an essential tumour antigen, but the lack of well-defined murine epitopes have delayed preclinical studies of Bcl-xL-targeting cancer vaccines. Here, we report the identification of two novel murine tumour-associated epitopes TAYQSFEQV and AFFSFGGAL derived from mouse Bcl-xL. Dendritic cell (DC)-based vaccination induced CD8(+) T cells capable of producing IFN-γ upon restimulation with these epitopes. Thus, our data may benefit the design of future immunotherapy strategies by providing a preclinical model for cancer vaccination with an endogenous tumour antigen that can be combined with other cancer treatments.

IgE versus IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy.

BACKGROUND: Development and maintenance of tolerance to food allergens appears to be associated with alterations in antigen specific IgE and IgG4 responses. Previous studies have focused only on comparing IgE and IgG4 linear epitope recognition patterns but take no account of conformational epitopes. OBJECTIVE: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. METHODS: Polyclonal sera from three individual patients, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library. Resulting epitope-mimicking sequences were aligned for identification of consensus sequences and localised on the surface of the Ara h 1 molecule by a computer-based algorithm. RESULTS: All epitope-mimicking sequences identified were found to correspond to conformational epitopes. Each individual patient had his/her own distinct IgE as well as IgG4 epitope recognition profile, though some important IgE epitopes were common to all patients. In general the IgG4 epitope pattern was more heterogeneous than the IgE pattern, did not coincide with IgE epitopes and had a lower affinity than IgE. CONCLUSIONS: This study demonstrated the usefulness of the phage-display technology in distinguishing between the epitope pattern of IgE and IgG4, giving detailed information on fine specificity and affinity. Competitive immuno-screening of phage-display random peptide libraries could be a future valuable tool to study the balance and dynamics of the IgE and IgG4 epitope recognition repertoire and provide a diagnostic tool giving information on the associated allergic phenotype.

Electron-nuclear energy sharing in above-threshold multiphoton dissociative ionization of H2.

We report experimental observation of the energy sharing between electron and nuclei in above-threshold multiphoton dissociative ionization of H2 by strong laser fields. The absorbed photon energy is shared between the ejected electron and nuclei in a correlated fashion, resulting in multiple diagonal lines in their joint energy spectrum governed by the energy conservation of all fragment particles.

Multiphoton above threshold effects in strong-field fragmentation.

We present a study of multiphoton dissociative ionization from molecules. By solving the time-dependent Schrödinger equation for H(2)(+) and projecting the solution onto double continuum scattering states, we observe the correlated electron-nuclear ionization dynamics in detail. We show-for the first time-how multiphoton structure prevails as long as one accounts for the energies of all the fragments. Our current work provides a new avenue to analyze strong-field fragmentation that leads to a deeper understanding of the correlated molecular dynamics.

IgE epitopes of intact and digested Ara h 1: a comparative study in humans and rats.

BACKGROUND: Allergen epitope characterization provides valuable information useful for the understanding of proteins as food allergens. It is believed that IgE epitopes in general are conformational, nevertheless, for food allergens known to sensitize through the gastrointestinal tract linear epitopes have been suggested to be of great importance. OBJECTIVE: The aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. METHODS: Sera from five peanut allergic patients and five Brown Norway rats were used to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule to mimic epitopes using a computer-based algorithm. RESULTS: Patients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which by far accounted for most of the eluted peptide sequences. Epitope patterns were rather similar for both intact and digested Ara h 1 as well as for humans and rats. CONCLUSIONS: Individual patient specific epitope patterns have been identified for the major allergen Ara h 1. IgE binding epitopes have been suggested as biomarkers for persistency and severity of food allergy, wherefore recognition of particular epitope patterns or motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergy.

Can we define a tolerable level of risk in food allergy? Report from a EuroPrevall/UK Food Standards Agency workshop.

BACKGROUND: There is an emerging consensus that, as with other risks in society, zero risk for food-allergic people is not a realistic or attainable option. Food allergy challenge data and new risk assessment methods offer the opportunity to develop quantitative limits for unintended allergenic ingredients which can be used in risk-based approaches. However, a prerequisite to their application is defining a tolerable level of risk. This requires a value judgement and is ultimately a 'societal' decision that has to involve all relevant stakeholders. OBJECTIVE: The aim of the workshop was to bring together key representatives from the stakeholders (regulators, food industry, clinical researchers and patients), and for the first time ever discuss the definition of a tolerable level of risk with regard to allergic reactions to food. RESULTS: The discussions revealed a consensus that zero risk was not a realistic option and that it is essential to address the current lack of agreed action levels for cross-contamination with allergens if food allergen management practice is to be improved. The discussions also indicated that it was difficult to define and quantify a tolerable level of risk, although both the clinical and the industry groups tried to do so. A consensus emerged that doing nothing was not a viable option, and there was a strong desire to take action to improve the current situation. CONCLUSIONS AND CLINICAL RELEVANCE: Two concrete actions were suggested: (1) Action levels should be derived from the data currently available. Different scenarios should be examined and further developed in an iterative process. On the basis of this work, a tolerable level of risk should be proposed. (2) 'One-dose' clinical trial with a low challenge dose should be performed in multiple centres to provide additional information about the general applicability of dose-distribution models and help validate the threshold levels derived.

Digested Ara h 1 has sensitizing capacity in Brown Norway rats.

BACKGROUND: Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion. OBJECTIVE: Using Ara h 1 as a model allergen, the ability of digested protein to sensitize has been investigated. METHODS: Ara h 1 was purified from whole roasted peanuts. Intact Ara h 1 was digested in an in vitro model, simulating the human gastrointestinal digestion process. Digestion products were analysed for peptide sizes and their ability to aggregate. Brown Norway (BN) rats, used as an animal model, were immunized with purified intact Ara h 1 or the gastrointestinal digestion products thereof. The sensitizing capacity was evaluated by analyses of specific antibody (IgG1, IgG2a and IgE) responses and ability to trigger mediator release of rat basophilic leukaemia (RBL)-2H3 cells. RESULTS: The present study showed that Ara h 1 was broken down, resulting in peptide fragments of sizes<2.0 kDa, of which approximately 50% was in aggregated complexes of Mr up to 20 kDa. Ara h 1 digesta were shown to have sensitizing capacity in BN rats, being capable of inducing specific IgG and IgE antibodies. The IgE response was functional, having the capacity to induce specific degranulation of RBL cells. CONCLUSION: From this study, it can be concluded that lability of a food allergen to gastrointestinal digestion does not necessarily abrogate its allergenic sensitizing potential.

A combined experimental and theoretical study on realizing and using laser controlled torsion of molecules.

It is demonstrated that strong laser pulses can introduce torsional motion in the axially chiral molecule 3,5-difluoro-3('),5(')-dibromobiphenyl. A nanosecond laser pulse spatially aligns the stereogenic carbon-carbon (C-C) bond axis allowing a perpendicularly polarized, intense femtosecond pulse to initiate torsional motion accompanied by a rotation about the fixed axis. We monitor the induced motion by femtosecond time-resolved Coulomb explosion imaging. Our theoretical analysis corroborates the experimental findings and on the basis of these results we discuss future applications of laser-induced torsion, viz., time-resolved studies of deracemization and laser controlled molecular junctions based on molecules with torsion.

Manipulating the torsion of molecules by strong laser pulses.

We demonstrate that strong laser pulses can induce torsional motion in a molecule consisting of a pair of phenyl rings. A nanosecond laser pulse spatially aligns the carbon-carbon bond axis, connecting the two phenyl rings, allowing a perpendicularly polarized, intense femtosecond pulse to initiate torsional motion accompanied by an overall rotation about the fixed axis. We monitor the induced motion by femtosecond time-resolved Coulomb explosion imaging. Our theoretical analysis accounts for and generalizes the experimental findings.

Approaches to risk assessment in food allergy: report from a workshop ''developing a framework for assessing the risk from allergenic foods".

A workshop was organised to investigate whether risk assessment strategies and methodologies used in classical/conventional toxicology may be used for risk assessment of allergenic foods, to discuss the advantages and limitations of different approaches and to determine the research needed to move the area forward. Three possible approaches to safety assessment and risk assessment for allergenic foods were presented and discussed: safety assessment using NOAEL/LOAEL and uncertainty factors, safety assessment using Benchmark Dose and Margin of Exposure (MoE), and risk assessment using probabilistic models. The workshop concluded that all the three approaches to safety and risk assessment of allergenic foods should continue to be considered. A particular strength of the MoE and probabilistic approaches is that they do not rely on low-dose extrapolations with its inherent issues. Probabilistic modelling is considered to be the most promising approach for use in population risk assessment (which is a particular focus for risk managers). For all approaches, further improvement of input data is desirable, particularly data on consumption patterns/food choices in food allergic consumers, data on minimum eliciting doses and data that can be used to evaluate whether the whole population at risk has been modelled accurately. Specific research topics were identified.

Treatment of cervical disc disease using Cloward's technique. Part V. The effect of reoperations.

Among 1,106 patients with cervical disc disease in whom Cloward's operation had been performed over a fifteen-year period, 145 patients were reoperated upon with a new Cloward procedure. In the 1st year after the first operation 3/4 of the patients were operated upon on the previous level, whereas reoperations performed later mainly were done on new levels. The effect of the reoperation was poorer than the effect of the 1st operation. Early reoperations--which were due to: an initially unsatisfactory clinical course, postoperative complications or graft problems--had a poor result, but late reoperations--which were caused by relapses--showed a result corresponding to the first operation. The frequency of relapses leading to a 2nd Cloward operation was 8.8% in the first year falling to 0.2% in the following years of observation.

The significance of preoperative radiological examinations in patients treated with Cloward's operation.

During a fifteen-year period 1,106 patients with symptoms and signs of cervical disc disease underwent Cloward's operation. Plain x-rays were performed in 94%, but the severity and extension of degenerative changes had no prognostic value. In 91% of the patients myelography was performed and in recent years mainly metrizamide was used as it was found to be more accurate. The findings on the myelograms were correlated according to age, severity and number of affected disc levels, but were independent of the duration of symptoms. Patients with medullary symptoms were found more often than not to have an anterior indentation into the spinal canal whereas most patients with radicular symptoms had wide cervical root sleeves or lateral compression. The best outcome was found among patients with monosegmental symptoms and signs and one affected disc level on the myelogram underlining that the indication for surgery in cervical disc disease should be based on both clinical and radiological findings.

Treatment of cervical disc disease using Cloward's technique. The prognostic value of clinical preoperative data in 1,106 patients.

During a fifteen-year period 1,106 patients underwent Cloward's operation due to cervical disc disease. An analysis of the clinical features showed that the diseases had a very complex pattern of symptoms and signs. In young patients a syndrome of herniated disc predominated whereas a clinical picture of "cervical spondylosis" was found more often in the elderly. It was demonstrated that best results were obtained in young patients, patients with slight symptoms and patients with monoradicular symptoms. A short duration of symptoms is essential for a good outcome, and, on the basis of the data presented, operation is recommended 3 months after the onset of symptoms of cervical disc disease, if previous conservative treatment has failed.

Treatment of cervical disc disease using Cloward's technique. III. Evaluation of cervical spondylotic myelopathy in 138 cases.

Among 1,106 patients with cervical disc disease treated with Cloward's operation over a fifteen-year period, 138 patients had symptoms and signs of spinal cord involvement. The median symptom duration was one year. Nearly all had gait disturbances, 63% had spasticity of the lower extremities and 11% had various degrees of tetraplegia. Three months after surgery 78% of the patients were found to have some symptoms, but only 5% had no symptoms at all. At the time of the questionnaire (average 7.7 years postoperatively) 46% of the patients stated they were still affected by the operation. The duration of preoperative symptoms was of significance to the outcome, but age, sex, and severity of myelopathy were not. It was demonstrated that patients with medullary involvement had a higher annual mortality rate than patients with radicular symptoms, and that this rate depended on the severity of the spinal cord symptoms.

Treatment of cervical disc disease using Cloward's technique. I. General results, effect of different operative methods and complications in 1,106 patients.

During the period from April, 1965, to October, 1979, 1,106 patients with symptoms or signs of cervical disc disease were operated on using Cloward's technique. 94% of the patients alive filled in a questionnaire forwarded on October, 1980. The study revealed that 81% experienced an immediate effect of the operation, and 63% were in a better condition than preoperatively at the time of the questionnaire. It appeared that the effect of the operation 1 year postoperatively was independent of age and observation time, but the functional result and postoperative working capacity were very significantly better in young patients, but still independent of observation time. The result was independent of the number of primarily inserted grafts, cutting of the posterior spinal ligament or the finding of a free herniated disc. Use of calf bone grafts resulted in more reoperations. Primary complications were registered in 13% of the patients, reduced to 5% after 3 months. 3 patients developed a severe tetraplegia, and 6 patients died within 3 months of the operation.

Radiumstationen in Aarhus from 1962 to 1984.

An overview is given of the development at Radiumstationen in Aarhus from 1962 to 1984. Within the field of radiation therapy the period has been characterized by the transition to megavoltage units, an extended use of dosimetry and dose planning, the clinical application of radiobiologic principles and the recent introduction of CT scanning for dose planning. The past ten years have seen a growing importance of chemotherapy and an intensified collaboration with the specialized branches of surgery, which among other things has brought about a change in the structure of the Department; a change towards sub-specialization according to the site of the organs treated. The period has also been characterized by an increasing number of patients with cancer and a very intensive utilization of wards and equipment, as well as by a busily engaged staff . The present function of the Department is that of Department of Oncology at one of the 5 Danish oncologic centres serving its specific region.