Systems Biology based studies on anti-inflammatory compounds

The introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in the life science area. In this thesis we explored the possibilities to apply these holistic technologies to investigate the effects of known and potential anti-inflammatory compounds on macrophages. For this purpose we made use of a monocyte-like human histocytic lymphoma cell line U937. U937 cells can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. The two differentiation stages, monocyte and macrophage, were compared by using oligonucleotide microarrays and 2-D gel electrophoresis in combination with principal component analysis (PCA). This differentiation study is described in Chapter 2. The differential expression of three protein biomarkers, gamma interferon inducible lysosomal thiol reductase (GILT), cathepsin D and adipocyte-fatty acid binding protein (A-FABP) were biologically validated by Western blot and real time polymerase chain reaction (real time PCR). GILT and A-FABP were also found to be differentially expressed at the mRNA level as indicated by the results of the microarray experiment. Moreover, the transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. From the results presented in Chapter 2 can be concluded that the U937 cell line is an excellent model system for the blood-derived macrophage and that microarrays and 2-D gel electrophoresis are suitable methods to identify biomarkers for differentiation. Chapter 3 describes the use of a systems biology approach to categorize anti-inflammatory drugs based on their mRNA, protein and lipid expression pattern, as determined by oligonucleotide microarrays, 2-D gel electrophoresis and a LC-MS method for lipids, in combination with principal component discriminant analysis (PC-DA). The results described in this chapter demonstrate that different classes of anti-inflammatory compounds show distinct and characteristic mRNA, protein, and lipid expression patterns, which can be used to categorize known anti-inflammatory drugs, as well as to discover and classify new leads. The latter was exemplified by the categorization of zilpaterol, a poorly characterized ____-agonist. Exposure to zilpaterol gives rise to an almost identical expression pattern as that observed after exposure to the well-characterized __2-agonists clenbuterol and salbutamol, suggesting that zilpaterol is indeed a ____-agonist. In addition, this study revealed potential biomarkers for the different anti-inflammatory drugs under investigation. The categorization of the anti-inflammatory drugs on the basis of proteomics data alone was not successful. The most likely explanation for this is that by the analysis of whole cell lysates, only highly abundant proteins can be visualized, while the low abundant proteins, which are often involved in important metabolic pathways, are not. Therefore, a more focused approach was used to investigate the mechanism of action of zilpaterol, which is described in Chapter 4. In Chapter 4, U937 macrophages were stimulated with LPS to induce an inflammatory response. This response was inhibited by the addition of zilpaterol (LZ) and this inhibition was antagonized by the _2-adrenergic receptor antagonist propranolol (LZP). Two-dimensional difference gel electrophoresis (DIGE) in combination with Student__s t-test and two multivariate data analysis tools (PCA and partial least squares discriminant analysis PLS-DA) were used to examine the secreted proteome induced by the three treatments. This revealed 8 potential protein biomarkers. The protein spots were identified using nano LC-MS-MS. Only two of the identified proteins, namely macrophage inflammatory protein-1_ (MIP-1_) and macrophage inflammatory protein-1_ (MIP-1_) are known to be secreted proteins. The inhibition of MIP-1_ by zilpaterol and the involvement of the _2-AR and cyclic adenosine-3__,5__-cyclic monophosphate (cAMP) were confirmed using a specific immuno-assay. The experiments described in this chapter demonstrate the importance of pre-fractionation of complex protein samples before performing proteomics studies. The categorization of zilpaterol in Chapter 3 as a _2-adrenegic receptor agonist was further explored in Chapter 5. In this chapter we investigated the binding affinity of zilpaterol to the _1- and _2 receptor by using a receptor binding assay. Furthermore, we examined the role of the _1- and _2 adrenoceptor in the inhibition of the LPS induced tumor necrosis factor-alpha (TNF-_) production and the induction of cAMP by U937 macrophages. For this purpose we made use of a selective _1-receptor antagonist (atenolol), a selective _2-antagonist (ICI 118551) and a non-selective _-antagonist (propranolol). Finally, the inhibitory effect of zilpaterol on the TNF-_ production was investigated in LPS-treated male Wistar rats. The results obtained in this way clearly show that zilpaterol is a _2-adrenergic agonist and a inhibitor of the LPS-induced TNF-_ production by macrophages both in vivo and in vitro. The three _2-agonists specific biomarkers, Granulocyte Chemotactic Protein-2 (GCP-2/CXCL6), Oncostatin M (OSM), and Vascular Endothelial Growth Factor (VEGF) that were identified in Chapter 3, were further examined in Chapter 6. The three markers were significantly up-regulated both in U937 macrophages and in blood-derived macrophages exposed to a _2-agonist (clenbuterol and zilpaterol) in the absence or presence of LPS, as determined by a specific enzyme-linked immunosorbent assays (ELISA). Moreover, this up-regulation was also accomplished by other cyclic AMP elevating agents (forskolin, prostaglandins E2, and dibutyryl cAMP), suggesting a role of cAMP in the up-regulation of GCP-2/CXCL6, VEGF and OSM. We hypothesize that these proteins may be involved in some of the adverse effects in the treatment of asthma with _2-adrenergic receptor agonists. In the second part of this thesis we focussed on a multi-component drug, namely Cannabis sativa. In Chapter 7, the immuno-modulating effects of unheated and heated Cannabis extracts were investigated. This study revealed that unheated Cannabis extracts and its major non-psychoactive compound _9-tetrahydrocannabinolic acid (THCa) were able to inhibit the LPS induced TNF-_ production both in U937 macrophages and in blood-derived macrophages. The inhibitory effect on TNF-_ was not mediated by the cannabinoid receptors CB1 and CB2. Furthermore, this study showed that unheated Cannabis extracts and THCa exert their inhibitory effect on the TNF-_ production via a mechanism that is different from that of heated Cannabis extract and its main constituent the psychoactive compound _9-tetrahydrocannabinol (THC). The inhibition of TNF-_ release by unheated Cannabis extract and THCa was prolonged over a relatively long period of time. By contrast, although THC and heated extracts initially inhibit the release of TNF-_, after longer incubation times they seem to increase TNF-_ production to levels that are even higher than in the absence of THC or Cannabis extract. This difference in response of the U937 macrophages to THC and THCa was also observed in an experiment in which we examined the effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibited the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. Finally, we studied the effect of THCa and unheated Cannabis extract in a pilot study using an Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Unheated Cannabis extract and THCa had a favourable effect on the clinical and histological signs of EAE. However, these results are preliminary and not clearly significant, therefore further investigation is necessary. Chapter 8 describes the categorization of unheated and heated Cannabis extracts using the same model system as described in Chapter 3. The mRNA patterns obtained from U937 macrophages exposed to LPS in the absence or presence of different anti-inflammatory drugs and unheated and heated Cannabis extracts were analysed using PC-DA. The study revealed that heated and unheated Cannabis extracts give rise to different expression patterns, which is in agreement with the observations made in Chapter 7 that they exert their TNF-_ inhibitory effect via different pathways. Moreover, their expression patterns did not overlap with that of other classes of anti-inflammatory compounds known to inhibit the TNF-_ production. These results suggest that the Cannabis extracts can not be assigned to one of the above mentioned classes of inflammatory inhibitors. Further investigation is necessary to unravel the exact mechanism of action of unheated and heated Cannabis extracts. In conclusion, the studies in this thesis show that the application of systems biology approaches are very useful in the categorization of anti-inflammatory compounds based on their mRNA and lipid expression patterns and to find specific biomarkers for these compounds. The categorization based on the protein expression pattern was less successful. This is most probably due to the fraction of proteins that was analysed on the gel. With proteomics techniques only a small fraction of proteins can be analysed simultaneously. Pre-fractionation, enrichment techniques and different analytical methods are therefore necessary to analyse a wide range of proteins with diverse physiological properties and dynamic range. The datasets obtained by transcriptomics, proteomics and metabolomics were analysed using statistical and pattern recognition tools. The datasets often contained a limited number of samples with respect to the large number of variables. It is therefore important to use these techniques as an explorative tool only and to validate the potential biomarkers found by additional individual measurements. Taken together, the use of systems biology for the investigation of anti-inflammatory drugs yielded very promising results, even though only a small part of the systems biology circle was used.TNO Quality of Life, Zeist, The Netherlands. Nonlinear Dynamics, Newcastle upon Tyne, UKUBL - phd migration 201

Food processing and allergenicity

Food processing can have many beneficial effects. However, processing may also alter the allergenic properties of food proteins. A wide variety of processing methods is available and their use depends largely on the food to be processed. In this review the impact of processing (heat and non-heat treatment) on the allergenic potential of proteins, and on the antigenic (IgG-binding) and allergenic (IgE-binding) properties of proteins has been considered. A variety of allergenic foods (peanuts, tree nuts, cows’ milk, hens’ eggs, soy, wheat and mustard) have been reviewed. The overall conclusion drawn is that processing does not completely abolish the allergenic potential of allergens. Currently, only fermentation and hydrolysis may have potential to reduce allergenicity to such an extent that symptoms will not be elicited, while other methods might be promising but need more data. Literature on the effect of processing on allergenic potential and the ability to induce sensitisation is scarce. This is an important issue since processing may impact on the ability of proteins to cause the acquisition of allergic sensitisation, and the subject should be a focus of future research. Also, there remains a need to develop robust and integrated methods for the risk assessment of food allergenicity

Immunological outcomes of allergen-specific immunotherapy in food allergy

IgE-mediated food allergies are caused by adverse immunologic responses to food proteins. Allergic reactions may present locally in different tissues such as skin, gastrointestinal and respiratory tract and may result is systemic life-threatening reactions. During the last decades, the prevalence of food allergies has significantly increased throughout the world, and considerable efforts have been made to develop curative therapies. Food allergen immunotherapy is a promising therapeutic approach for food allergies that is based on the administration of increasing doses of culprit food extracts, or purified, and sometime modified food allergens. Different routes of administration for food allergen immunotherapy including oral, sublingual, epicutaneous and subcutaneous regimens are being evaluated. Although a wealth of data from clinical food allergen immunotherapy trials has been obtained, a lack of consistency in assessed clinical and immunological outcome measures presents a major hurdle for evaluating these new treatments. Coordinated efforts are needed to establish standardized outcome measures to be applied in food allergy immunotherapy studies, allowing for better harmonization of data and setting the standards for the future research. Several immunological parameters have been measured in food allergen immunotherapy, including allergen-specific immunoglobulin levels, basophil activation, cytokines, and other soluble biomarkers, T cell and B cell responses and skin prick tests. In this review we discuss different immunological parameters and assess their applicability as potential outcome measures for food allergen immunotherapy that may be included in such a standardized set of outcome measures

Metabolic Effects of n-3 PUFA as Phospholipids Are Superior to Triglycerides in Mice Fed a High-Fat Diet: Possible Role of Endocannabinoids

Background n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides. Methodology/Principal Findings In a ‘prevention study’, C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ~35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced 2-arachidonoylglycerol, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine. Conclusions/Significance Compared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WA

Systems Biology based studies on anti-inflammatory compounds

The introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in the life science area. In this thesis we explored the possibilities to apply these holistic technologies to investigate the effects of known and potential anti-inflammatory compounds on macrophages. For this purpose we made use of a monocyte-like human histocytic lymphoma cell line U937. U937 cells can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. The two differentiation stages, monocyte and macrophage, were compared by using oligonucleotide microarrays and 2-D gel electrophoresis in combination with principal component analysis (PCA). This differentiation study is described in Chapter 2. The differential expression of three protein biomarkers, gamma interferon inducible lysosomal thiol reductase (GILT), cathepsin D and adipocyte-fatty acid binding protein (A-FABP) were biologically validated by Western blot and real time polymerase chain reaction (real time PCR). GILT and A-FABP were also found to be differentially expressed at the mRNA level as indicated by the results of the microarray experiment. Moreover, the transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. From the results presented in Chapter 2 can be concluded that the U937 cell line is an excellent model system for the blood-derived macrophage and that microarrays and 2-D gel electrophoresis are suitable methods to identify biomarkers for differentiation. Chapter 3 describes the use of a systems biology approach to categorize anti-inflammatory drugs based on their mRNA, protein and lipid expression pattern, as determined by oligonucleotide microarrays, 2-D gel electrophoresis and a LC-MS method for lipids, in combination with principal component discriminant analysis (PC-DA). The results described in this chapter demonstrate that different classes of anti-inflammatory compounds show distinct and characteristic mRNA, protein, and lipid expression patterns, which can be used to categorize known anti-inflammatory drugs, as well as to discover and classify new leads. The latter was exemplified by the categorization of zilpaterol, a poorly characterized ____-agonist. Exposure to zilpaterol gives rise to an almost identical expression pattern as that observed after exposure to the well-characterized __2-agonists clenbuterol and salbutamol, suggesting that zilpaterol is indeed a ____-agonist. In addition, this study revealed potential biomarkers for the different anti-inflammatory drugs under investigation. The categorization of the anti-inflammatory drugs on the basis of proteomics data alone was not successful. The most likely explanation for this is that by the analysis of whole cell lysates, only highly abundant proteins can be visualized, while the low abundant proteins, which are often involved in important metabolic pathways, are not. Therefore, a more focused approach was used to investigate the mechanism of action of zilpaterol, which is described in Chapter 4. In Chapter 4, U937 macrophages were stimulated with LPS to induce an inflammatory response. This response was inhibited by the addition of zilpaterol (LZ) and this inhibition was antagonized by the _2-adrenergic receptor antagonist propranolol (LZP). Two-dimensional difference gel electrophoresis (DIGE) in combination with Student__s t-test and two multivariate data analysis tools (PCA and partial least squares discriminant analysis PLS-DA) were used to examine the secreted proteome induced by the three treatments. This revealed 8 potential protein biomarkers. The protein spots were identified using nano LC-MS-MS. Only two of the identified proteins, namely macrophage inflammatory protein-1_ (MIP-1_) and macrophage inflammatory protein-1_ (MIP-1_) are known to be secreted proteins. The inhibition of MIP-1_ by zilpaterol and the involvement of the _2-AR and cyclic adenosine-3__,5__-cyclic monophosphate (cAMP) were confirmed using a specific immuno-assay. The experiments described in this chapter demonstrate the importance of pre-fractionation of complex protein samples before performing proteomics studies. The categorization of zilpaterol in Chapter 3 as a _2-adrenegic receptor agonist was further explored in Chapter 5. In this chapter we investigated the binding affinity of zilpaterol to the _1- and _2 receptor by using a receptor binding assay. Furthermore, we examined the role of the _1- and _2 adrenoceptor in the inhibition of the LPS induced tumor necrosis factor-alpha (TNF-_) production and the induction of cAMP by U937 macrophages. For this purpose we made use of a selective _1-receptor antagonist (atenolol), a selective _2-antagonist (ICI 118551) and a non-selective _-antagonist (propranolol). Finally, the inhibitory effect of zilpaterol on the TNF-_ production was investigated in LPS-treated male Wistar rats. The results obtained in this way clearly show that zilpaterol is a _2-adrenergic agonist and a inhibitor of the LPS-induced TNF-_ production by macrophages both in vivo and in vitro. The three _2-agonists specific biomarkers, Granulocyte Chemotactic Protein-2 (GCP-2/CXCL6), Oncostatin M (OSM), and Vascular Endothelial Growth Factor (VEGF) that were identified in Chapter 3, were further examined in Chapter 6. The three markers were significantly up-regulated both in U937 macrophages and in blood-derived macrophages exposed to a _2-agonist (clenbuterol and zilpaterol) in the absence or presence of LPS, as determined by a specific enzyme-linked immunosorbent assays (ELISA). Moreover, this up-regulation was also accomplished by other cyclic AMP elevating agents (forskolin, prostaglandins E2, and dibutyryl cAMP), suggesting a role of cAMP in the up-regulation of GCP-2/CXCL6, VEGF and OSM. We hypothesize that these proteins may be involved in some of the adverse effects in the treatment of asthma with _2-adrenergic receptor agonists. In the second part of this thesis we focussed on a multi-component drug, namely Cannabis sativa. In Chapter 7, the immuno-modulating effects of unheated and heated Cannabis extracts were investigated. This study revealed that unheated Cannabis extracts and its major non-psychoactive compound _9-tetrahydrocannabinolic acid (THCa) were able to inhibit the LPS induced TNF-_ production both in U937 macrophages and in blood-derived macrophages. The inhibitory effect on TNF-_ was not mediated by the cannabinoid receptors CB1 and CB2. Furthermore, this study showed that unheated Cannabis extracts and THCa exert their inhibitory effect on the TNF-_ production via a mechanism that is different from that of heated Cannabis extract and its main constituent the psychoactive compound _9-tetrahydrocannabinol (THC). The inhibition of TNF-_ release by unheated Cannabis extract and THCa was prolonged over a relatively long period of time. By contrast, although THC and heated extracts initially inhibit the release of TNF-_, after longer incubation times they seem to increase TNF-_ production to levels that are even higher than in the absence of THC or Cannabis extract. This difference in response of the U937 macrophages to THC and THCa was also observed in an experiment in which we examined the effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibited the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. Finally, we studied the effect of THCa and unheated Cannabis extract in a pilot study using an Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Unheated Cannabis extract and THCa had a favourable effect on the clinical and histological signs of EAE. However, these results are preliminary and not clearly significant, therefore further investigation is necessary. Chapter 8 describes the categorization of unheated and heated Cannabis extracts using the same model system as described in Chapter 3. The mRNA patterns obtained from U937 macrophages exposed to LPS in the absence or presence of different anti-inflammatory drugs and unheated and heated Cannabis extracts were analysed using PC-DA. The study revealed that heated and unheated Cannabis extracts give rise to different expression patterns, which is in agreement with the observations made in Chapter 7 that they exert their TNF-_ inhibitory effect via different pathways. Moreover, their expression patterns did not overlap with that of other classes of anti-inflammatory compounds known to inhibit the TNF-_ production. These results suggest that the Cannabis extracts can not be assigned to one of the above mentioned classes of inflammatory inhibitors. Further investigation is necessary to unravel the exact mechanism of action of unheated and heated Cannabis extracts. In conclusion, the studies in this thesis show that the application of systems biology approaches are very useful in the categorization of anti-inflammatory compounds based on their mRNA and lipid expression patterns and to find specific biomarkers for these compounds. The categorization based on the protein expression pattern was less successful. This is most probably due to the fraction of proteins that was analysed on the gel. With proteomics techniques only a small fraction of proteins can be analysed simultaneously. Pre-fractionation, enrichment techniques and different analytical methods are therefore necessary to analyse a wide range of proteins with diverse physiological properties and dynamic range. The datasets obtained by transcriptomics, proteomics and metabolomics were analysed using statistical and pattern recognition tools. The datasets often contained a limited number of samples with respect to the large number of variables. It is therefore important to use these techniques as an explorative tool only and to validate the potential biomarkers found by additional individual measurements. Taken together, the use of systems biology for the investigation of anti-inflammatory drugs yielded very promising results, even though only a small part of the systems biology circle was used.</p

Insect (food) allergy and allergens

Insects represent an alternative for meat and fish in satisfying the increasing demand for sustainable sources of nutrition. Approximately two billion people globally consume insects. They are particularly popular in Asia, Latin America, and Africa. Most research on insect allergy has focussed on occupational or inhalation allergy. Research on insect food safety, including allergenicity, is therefore of great importance. The objective of this review is to provide an overview of cases reporting allergy following insect ingestion, studies on food allergy to insects, proteins involved in insect allergy including cross-reactive proteins, and the possibility to alter the allergenic potential of insects by food processing and digestion. Food allergy to insects has been described for silkworm, mealworm, caterpillars, Bruchus lentis, sago worm, locust, grasshopper, cicada, bee, Clanis bilineata, and the food additive carmine, which is derived from female Dactylopius coccus insects. For cockroaches, which are also edible insects, only studies on inhalation allergy have been described. Various insect allergens have been identified including tropomyosin and arginine kinase, which are both pan-allergens known for their cross-reactivity with homologous proteins in crustaceans and house dust mite. Cross-reactivity and/or co-sensitization of insect tropomyosin and arginine kinase has been demonstrated in house dust mite and seafood (e.g. prawn, shrimp) allergic patients. In addition, many other (allergenic) species (various non-edible insects, arachnids, mites, seafoods, mammals, nematoda, trematoda, plants, and fungi) have been identified with sequence alignment analysis to show potential cross-reactivity with allergens of edible insects. It was also shown that thermal processing and digestion did not eliminate insect protein allergenicity. Although purified natural allergens are scarce and yields are low, recombinant allergens from cockroach, silkworm, and Indian mealmoth are readily available, giving opportunities for future research on diagnostic allergy tests and vaccine candidates

Allergenicity assessment strategy for novel food proteins and protein sources

To solve the future food insecurity problem, alternative and sustainable protein sources (e.g. insects, rapeseed, fava bean and algae) are now being explored for the production of food and feed. To approve these novel protein sources for future food a comprehensive risk assessment is needed according to the European food legislation. Allergenicity risk assessment might pose some major difficulties, since detailed guidance on how to assess the allergenic potential of novel foods is not available. At present, the approach relies mostly on the guidance of allergenicity assessment for genetically modified (GM) plant foods. The most recent one was proposed by EFSA (2010 and 2011); "weight-of-evidence approach". However this guidance is difficult to interpret, not completely applicable or validated for novel foods and therefore needs some adjustments. In this paper we propose a conceptual strategy which is based on the "weight-of-evidence approach" for food derived from GM plants and other strategies that were previously published in the literature. This strategy will give more guidance on how to assess the allergenicity of novel food proteins and protein sources

Allergenicity assessment strategy for novel food proteins and protein sources

To solve the future food insecurity problem, alternative and sustainable protein sources (e.g. insects, rapeseed, fava bean and algae) are now being explored for the production of food and feed. To approve these novel protein sources for future food a comprehensive risk assessment is needed according to the European food legislation. Allergenicity risk assessment might pose some major difficulties, since detailed guidance on how to assess the allergenic potential of novel foods is not available. At present, the approach relies mostly on the guidance of allergenicity assessment for genetically modified (GM) plant foods. The most recent one was proposed by EFSA (2010 and 2011); "weight-of-evidence approach". However this guidance is difficult to interpret, not completely applicable or validated for novel foods and therefore needs some adjustments. In this paper we propose a conceptual strategy which is based on the "weight-of-evidence approach" for food derived from GM plants and other strategies that were previously published in the literature. This strategy will give more guidance on how to assess the allergenicity of novel food proteins and protein sources

Legume Protein Consumption and the Prevalence of Legume Sensitization

Sensitization and allergy to legumes can be influenced by different factors, such as exposure, geographical background, and food processing. Sensitization and the allergic response to legumes differs considerably, however, the reason behind this is not yet fully understood. The aim of this study is to investigate if there is a correlation between legume protein consumption and the prevalence of legume sensitization. Furthermore, the association between sensitization to specific peanut allergens and their concentration in peanut is investigated. Legume sensitization data (peanut, soybean, lupin, lentil, and pea) from studies were analyzed in relation to consumption data obtained from national food consumption surveys using the European Food Safety Authority (EFSA), Global Environment Monitoring System (GEMS), and What We Eat in America&mdash;Food Commodity Intake Database (WWEIA-FCID) databases. Data were stratified for children &lt;4 years, children 4&ndash;18 years, and adults. Sufficient data were available for peanut to allow for statistical analysis. Analysis of all age groups together resulted in a low correlation between peanut sensitization and relative peanut consumption (r = 0.407), absolute peanut consumption (r = 0.468), and percentage of peanut consumers (r = 0.243). No correlation was found between relative concentrations of Ara h 1, 2, 3, 6, 7, and 8 in peanut and sensitization to these peanut allergens. The results indicate that the amount of consumption only plays a minor role in the prevalence of sensitization to peanut. Other factors, such as the intrinsic properties of the different proteins, processing, matrix, frequency, timing and route of exposure, and patient factors might play a more substantial role in the prevalence of peanut sensitization

Legume Protein Consumption and the Prevalence of Legume Sensitization

Sensitization and allergy to legumes can be influenced by different factors, such as exposure, geographical background, and food processing. Sensitization and the allergic response to legumes differs considerably, however, the reason behind this is not yet fully understood. The aim of this study is to investigate if there is a correlation between legume protein consumption and the prevalence of legume sensitization. Furthermore, the association between sensitization to specific peanut allergens and their concentration in peanut is investigated. Legume sensitization data (peanut, soybean, lupin, lentil, and pea) from studies were analyzed in relation to consumption data obtained from national food consumption surveys using the European Food Safety Authority (EFSA), Global Environment Monitoring System (GEMS), and What We Eat in America-Food Commodity Intake Database (WWEIA-FCID) databases. Data were stratified for children <4 years, children 4⁻18 years, and adults. Sufficient data were available for peanut to allow for statistical analysis. Analysis of all age groups together resulted in a low correlation between peanut sensitization and relative peanut consumption (r = 0.407), absolute peanut consumption (r = 0.468), and percentage of peanut consumers (r = 0.243). No correlation was found between relative concentrations of Ara h 1, 2, 3, 6, 7, and 8 in peanut and sensitization to these peanut allergens. The results indicate that the amount of consumption only plays a minor role in the prevalence of sensitization to peanut. Other factors, such as the intrinsic properties of the different proteins, processing, matrix, frequency, timing and route of exposure, and patient factors might play a more substantial role in the prevalence of peanut sensitization