Vinken, M., Doktorova, T., Ellinger-Ziegelbauer, H., Ahr, H.-J., Lock, E., Carmichael, P., Roggen, E., van Delft, J., Kleinjans, J., Castell, J., Bort, R., Donato, T., Ryan, M., Corvi, R., Keun, H., Ebbels, T., Athersuch, T., Sansone, S.-A., Rocca-Serra, P., Stierum, R., Jennings, P., Pfaller, W., Gmuender, H., Vanhaecke, T., Rogiers, V.
"The carcinoGENOMICS project: Critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays"
Mutation Research-Reviews in Mutation Research, . Article in Press. (2008)
Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable. © 2008.
Devin, M., Mortellaro, S.
"Catering for an animal-free culture"
Manufacturing Chemist, 79 (3), pp. 27-28. (2008)
Larsen, A.I., Johnsen, C.R., Frickmann, J., Mikkelsen, S.
"Incidence of respiratory sensitisation and allergy to enzymes among employees in an enzyme producing plant and the relation to exposure and host factors"
Occupational and Environmental Medicine, 64 (11), pp. 763-768. (2007)
Objectives: Belonging to the group of high molecular weight respiratory sensitisers, microbial enzymes have been reported as a well known cause of occupational allergy, typically manifesting itself as rhinitis and/or asthma. High exposure to such high molecular weight sensitisers, and possibly also peak exposures, implies a higher risk than low exposure, but the exact relation between exposure, sensitisation and clinical allergy remains to be clarified. The authors sought to estimate the risk of respiratory enzyme allergy in an enzyme producing plant and to assess the relation between exposure indices and allergy. Methods: Retrospective follow-up study based upon data gathered from health surveillance since 1970. 1207 employees from production and laboratories were included. The level of enzyme exposure in the relevant departments was estimated retrospectively into five exposure levels based on 10-fold increments/decrements of the threshold limit value and other exposure information. The risk was estimated in an exponential regression survival model fitted with constant intensity for subperiods of time using maximum likelihood estimation. Results: During the first three years of a person's employment, the enzyme sensitisation and allergy incidence rates were 0.13 and 0.03 per person-year at risk, respectively. In the fitted models, exposure class did not correlate with the outcome variables. The risk of sensitisation decreased along the three decades, whereas the risk of allergy remained unchanged. The risk of sensitisation and allergy was doubled among smokers. Pre-employment atopy was only associated with sensitisation risk. Conclusion: Sensitisation to enzymes decreased during the study period, possibly reflecting improvements in the working environment. A similar decrease could not be demonstrated for allergy to enzymes. Neither of the two outcomes correlated with exposure estimates, possibly because of the low precision of the estimates.
Rovida, C., Basketter, D., Casati, S., De Silva, O., Hermans, H., Kimber, I., Manou, I., Weltzien, H.U., Roggen, E.
"Management of an integrated project (Sens-it-iv) to develop in vitro tests to assess sensitisation"
ATLA Alternatives to Laboratory Animals, 35 (3), pp. 317-322. (2007)
Sens-it-iv is an integrated project, funded by European Commission Framework Programme 6, the overall objective of which is to develop in vitro tests and test strategies to be used by the chemical, cosmetic and pharmaceutical industries to assess the risk for potential contact and respiratory sensitisers. Such tests, once formally validated and accepted, will permit the evaluation of the sensitising potential of existing and new chemical entities and the products of the European industries for classification and labelling, as required by the new EU REACH legislation on chemicals, or for the purpose of risk assessment as required by the 7th Amendment to the EU Cosmetics Directive. Sens-it-iv involves 28 partners, representing industries, universities and regulatory bodies, including various institutes in the EU Member States and different competencies, all with the common aim of achieving a final deliverable - increasing the safety of consumer products, whilst reducing animal experimentation. This paper provides an overview of the structure of the project and a detailed description of the organisation of its management.
Mortellaro, S., Devine, M.
"Advances in animal-free manufacturing of biopharmaceuticals"
BioPharm International, 20 (5 SUPPL.), pp. 30-37. (2007)
Kimber, I., Agius, R., Basketter, D.A., Corsini, E., Cullinan, P., Dearman, R.J., Gimenez-Arnau, E., Greenwell, L., Hartung, T., Kuper, F., Maestrelli, P., Roggen, E., Rovida, C., Casati, S.
"Chemical respiratory allergy: Opportunities for hazard identification and characterisation"
ATLA Alternatives to Laboratory Animals, 35 (2), pp. 243-265. (2007)
Basketter, D., Pease, C., Kasting, G., Kimber, I., Casati, S., Cronin, M., Diembeck, W., Gerberick, F., Hadgraft, J., Hartung, T., Marty, J.P., Nikolaidis, E., Patlewicz, G., Roberts, D., Roggen, E., Rovida, C., Van De Sandt, J.
"Skin sensitisation and epidermal disposition: The relevance of epidermal disposition for sensitisation hazard identification and risk assessment: The report and recommendations of ECVAM workshop 59a"
ATLA Alternatives to Laboratory Animals, 35 (1), pp. 137-154. (2007)
Roggen, E.L., Soni, N.K., Verheyen, G.R.
"Respiratory immunotoxicity: An in vitro assessment"
Toxicology in Vitro, 20 (8), pp. 1249-1264. (2006)
As yet, in vitro assessment of the immunotoxic potency of respiratory agents is not possible. The complexity of the endpoint and the respiratory tract, and the limited availability of well-documented respiratory agents are the main reasons. The evidence that epithelial cells (ECs) are triggered by compounds to express in vitro surface proteins and soluble mediators, has stimulated their use for developing tests for respiratory immunotoxicity. A variety of airway ECs and EC-lines have been assessed, but the available information seems to point at human alveolar cells (e.g., A549) as the most convenient cell type. EC-based test formats with various degrees of complexity have been assessed. Sofar, promising results were obtained using a 3D model using the human A549 lung cell line. Dendritic cells (DCs) have been subjected to intensive research. However, currently available tests are not well suited to discern among the potency of sensitizers. Potential explanations include the lack of standardised protocols for the generation of DCs, no good standards for estimating the quality of in vitro derived DC-cultures, and limited dynamics of the currently used end-points. Alveolar macrophages (AMs) have sofar received less attention. This may proof unjustified as macrophages may link innate responses to adaptive immunity. The observation that ECs, DCs and AMs affect each other, suggests that test formats are required combining at least two of these cell types if ranking of compounds according to their sensitising potency is the aim. In addition, the capacity of compounds to cross a cellular membrane is an important property of an immunotoxic compound, which can be assessed only in 3D reconstituted human tissue models. While promising data have been reported for the skin, immunocompetent 3D reconstituted human lung remains to be evaluated for respiratory immunotoxicity. Obviously, the success of any of these simplified test (as compared to the complexity of the immune response) is highly dependent on the availability of early stage biomarkers (expressed at mucosal barrier level) that are predictive for relevant immunotoxicity mechanisms occurring down-stream of the immune response. As yet, such biomarkers are not yet available. © 2006 Elsevier Ltd. All rights reserved.
Bindslev-Jensen, C., Skov, P.S., Roggen, E.L., Hvass, P., Brinch, D.S.
"Investigation on possible allergenicity of 19 different commercial enzymes used in the food industry"
Food and Chemical Toxicology, 44 (11), pp. 1909-1915. (2006)
The aim of the study was to investigate the safety to allergic patients of 19 commercially available and authority-approved enzymes used in the food industry. Enzymes produced by genetically modified organisms were included. Four hundred consecutive adult patients with a diagnosed allergy to inhalation allergens, food allergens, bee or wasp were included. All had at least one positive skin prick test to the above allergens. Skin prick testing with the 19 enzymes was performed on the forearm and if positive (in 13 patients), in vitro histamine release from blood basophils were performed. Patients with positive results in skin prick test were subsequently reinvestigated with further purified enzymes and finally challenged orally with the enzymes in a double-blind, placebo-controlled protocol. Only one reaction to a placebo challenge was seen. In some instances a positive skin prick test result or a positive histamine release was seen elicited by the enzymes, but since none of the patients were positive to any of the commercial enzymes in the subsequent oral challenges using exaggerated dosages of the enzymes compared to normal daily intake, the findings are without clinical relevance. A wide variety of enzyme classes and origins was included in the study. Because there were no allergenic findings of clinical relevance it is concluded that ingestion of food enzymes in general is not considered to be a concern with regard to food allergy. © 2006 Elsevier Ltd. All rights reserved.
Mittag, D., Batori, V., Neudecker, P., Wiche, R., Friis, E.P., Ballmer-Weber, B.K., Vieths, S., Roggen, E.L.
"A novel approach for investigation of specific and cross-reactive IgE epitopes on Bet v 1 and homologous food allergens in individual patients"
Molecular Immunology, 43 (3), pp. 268-278. (2006)
Background: A clinically relevant allergic reaction requires recognition of at least two different epitopes on the surface of the allergen by IgE. These epitopes may be specific or cross-reactive. Moreover, patterns of IgE reactivity may be patient-specific. The aim of our study was to compare specific and cross-reactive IgE epitopes and epitope patterns between individual patients. We used Bet v 1-related food allergy as a model. Methods: Five patients were investigated by cross-competitive ELISA for specific and cross-reacting IgE to Bet v 1, and its homologues Gly m 4 (soybean), Ara h 8 (peanut), and Pru av 1 (cherry). Allergen-specific as well as cross-reactive IgE epitopes were assessed by competitive immunoscreening of a phage-displayed random 7-mer peptide library using polyclonal purified IgE from individual sera. The resulting peptide mimics were mapped on the surface of the 3D-structure of the allergens using a computer-based algorithm. Results: Competitive immunoscreening and epitope mapping identified patient-specific IgE epitope patterns. However, one IgE-binding surface area that was recognized by all patients and two recognized by three patients were identified on all four proteins. These results are consistent with the determination of IgE cross-reactivity of the individual patients' sera against the four recombinant allergens by cross-competitive ELISA. Conclusions: Selection of phage-displayed peptide mimics with serum IgE from allergic patients in combination with computer-based mapping of the peptide mimics onto the surface of the three-dimensional allergen structure is a promising novel tool to investigate IgE epitope specificity in individual patients. Such basic information on epitope structure may be used for prediction of cross-reactivity and potential allergenicity of novel foods. © 2005 Elsevier Ltd. All rights reserved.
Batori, V., Friis, E.P., Nielsen, H., Roggen, E.L.
"An in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context"
Journal of Molecular Recognition, 19 (1), pp. 21-29. (2006)
Presently X-ray crystallography of protein-antibody complexes is still the most direct way of identifying B-cell epitopes. The objective of this study was to assess the potential of a computer-based epitope mapping tool (EMT) using antigenic amino acid motifs as a fast alternative in a number of applications not requiring detailed information, e.g. development of pharmaceutical proteins, vaccines and industrial enzymes. Using Gal d 4 as a model protein, the EMT was capable of identifying, in the context of the folded protein, amino acid positions known to be involved in antibody binding. The high sensitivity and positive predictive value of the EMT as well as the relevance of the structural associations suggested by the EMT indicated the existence of amino acid motifs that are likely to be involved in antigenic determinants. In addition, differential mapping revealed that sensitivity and positive predictive value were dependent on the minimum relative surface accessibility (RSA) of the amino acids included in the mapping, demonstrating that the EMTs accommodated for the fact that epitopes are three-dimensional entities with various degrees of accessibility. The comparison with existing prediction scales demonstrated the superiority of the EMT with respect to physico-chemical scales. The mapping tool also performed better than the available structural scales, but the significance of the differences remains to be established. Thus, the EMT has the potential of becoming a fast and simple alternative to X-ray crystallography for predicting structural antigenic determinants, if detailed epitope information is not required. Copyright © 2005 John Wiley & Sons, Ltd.
Lindstedt, M., Schiött, Å., Johnsen, C.R., Roggen, E., Johansson-Lindbom, B., Borrebaeck, C.A.K.
"Individuals with occupational allergy to detergent enzymes display a differential transcriptional regulation and cellular immune response"
Clinical and Experimental Allergy, 35 (2), pp. 199-206. (2005)
Background: In spite of significant safety measures, allergy to industrial enzymes remains a major concern. The increasing prevalence of occupational allergy emphasizes the need to investigate the functional properties of enzyme-exposed dendritic cells (DCs), as DCs possess a potent ability to activate allergen-specific T cells. Objective: This study aims at elucidating the molecular mechanisms underlying allergic immune responses to lipase, an industrial enzyme. For this purpose, we studied the effect of both hypoallergenic and wild-type lipase on the transcriptional regulation in DCs and their stimulatory effect on memory CD4+ T cells. Methods: Five individuals with documented lipase allergy were tested for specific serum IgE. DCs from these individuals, stimulated with lipases, were assayed for their ability to affect proliferation and polarization of memory T cells. The effect of lipases on transcriptional activity in DCs was evaluated using global expression analysis. Results: Lipase-specific IgE levels varied considerably between donors, with donor 4 exhibiting highest levels, and a potent specific CD4+ T cell recall response was demonstrated only for donor 4. No difference was detected in cytokine profile when T cells from donor 4 were co-cultured with DCs pulsed with either hypoallergenic or wild-type lipase, as demonstrated by high IL-4 and IL-13, and low IFN-γ production. However, the lipases induced different genetic signatures in DCs from donor 4, as compared with the non-responders. Conclusions: DCs from individuals with clinically diagnosed allergy to lipase displayed a differential response to stimulation with hypoallergenic and wild-type lipase in vitro. Only allergen-pulsed DCs from donor 4 were able to induce CD4+ T cell proliferation. The lipase-specific T cells displayed a T-helper type 2 phenotype, which was not altered by hypoallergenic lipase-pulsed DCs. Furthermore, DCs derived from donor 4 and stimulated with either of the lipases displayed different transcriptional profiles, as compared with the other donors. These signatures represent genes of potential importance for an immunoregulatory role of DC in an ongoing allergic response. © 2005 Blackwell Publishing Ltd.
Jakobsen, C.G., Bodtger, U., Kristensen, P., Poulsen, L.K., Roggen, E.L.
"Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries"
Molecular Immunology, 41 (10), pp. 941-953. (2004)
Allergen-specific Fab fragments isolated from combinatorial IgE and IgG libraries are useful tools for studying allergen-antibody interactions. To characterise the interaction between different allergens and antibodies we have created recombinant human phage antibody libraries in the Fab format. Human IgE and IgG libraries have been created from patients allergic to birch pollen or lipase. These libraries have been used to select binders recognising the major birch pollen allergen Bet v 1 and Humicola lanuginosa lipase. A panel of allergen-specific IgE and IgG antibodies were identified; these were further characterised by allergen binding studies using Biacore and competition studies using human sera and antibodies purified from human sera. Affinities in the nM range were recorded and a competition with human sera for allergen binding was observed. © 2004 Elsevier Ltd. All rights reserved.
Ahmad, S.K., Brinch, D.S., Friis, E.P., Pedersen, P.B.
"Toxicological studies on lactose oxidase from microdochium nivale expressed in Fusarium venenatum"
Regulatory Toxicology and Pharmacology, 39 (3), pp. 256-270. (2004)
A new carbohydrate oxidase, Lactose Oxidase, with high specificity of oxidizing the disaccharide lactose to lactobionic acid has been found. This enzyme opens up for a variety of applications. A programme of toxicological studies was conducted to establish the safety of Lactose Oxidase to be used as a processing aid in the food industry. The enzyme used in this study was produced by a submerged fermentation of Fusarium venenatum and contained a gene code from Microdochium nivale. Oral administration to rats of up to 10mL/kg bodyweight (bw)/day (equivalent to a total organic solids dosage of 900mg/kgbw/day or a Lactose Oxidase dosage of 344LOXU/kgbw/day) for 13 weeks did not cause any adverse effect. Lactose Oxidase was not found to be mutagenic in the bacterial reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes. The maximum recommended dosage of Lactose Oxidase is 50LOXU/kg liquid whey protein concentrate. The safety margin for exposure is estimated to be at least 6.2×104 for daily diary product consumption. In conclusion Lactose Oxidase can be considered as safe for use in the food industry. © 2003 Elsevier Inc. All rights reserved.
J.T. Andersen; T. Schafer; P.L Jørgensen; S. Møller.
"Using inactivated microbial biomass as fertilizer: The fate of antibiotic resistance genes in the environment."
Research in Microbiology, 152(9), 823-833 (2001)
The waste product produced by Novo Nordisk A/S from microbial fermentations is used as agricultural fertilizer in Denmark (NovoGro (TM)) after being treated by heat and chemicals to destroy the microorganisms. The fertilizer contains DNA fragments from the genetically modified microorganisms used in industrial production. This DNA contains genes coding for the desired industrial products as well as genes used as genetic selection markers during production strain development. The antibiotic resistance markers used as genetic selection markers are chloramphenicol (Cm), kanamycin (Km) and ampicillin (Ap). The aim of the present study Was to examine whether DNA and intact genes were present in NovoGro and whether horizontal transfer of DNA isolated from inactivated production strains occurred either in the laboratory or in the fields treated with NovoGro. DNA isolated from NovoGro was analysed by PCR and intact genes coding for a protease and chloramphenicol resistance were amplified. This isolated DNA was used for in vitro experiments including electroporation and transformation but no transfer of DNA to Escherichia coli or Bacillus subtilis was observed. The antibiotic resistance profile of the indigenous bacterial population in the fields treated with NovoGro compared with fields treated with inorganic fertilizers showed no differences. In addition, DNA isolated directly from the fields treated with NovoGro for up to 7 years was analysed by PCR and no specific production gene constructs could be detected.