All days

Experimental Plan

Experimental plan

WP5a Human Nutrition

Many bioactive molecules found in foods of plant origin (including phenolic compounds, methyltocols and carotenoids) of which apples are especially rich, are able to interact with human cells and modulate its physiopathological processes. This interaction takes place in different ways, amongst which the antioxidant mechanisms are well-known, but more specific interactions which impact on cell signalling have been described resulting in gene expression modulation with substantial positive effects on degenerative diseases, such as cancer and cardiomyopathies. The therapeutic exploitation of these molecules is conditioned by their bioavailability and by complex transformation processes taking place after their ingestion. The networks (WP1, 2 and 3) will bring in their expertise to define ideotypical composition ensuring an optimal combination of the nutritional properties of apples.

The research will be carried out through the following initiatives: i) study of biotransformation and metabolism of apple compounds with the identification of the most bioactive forms, ii) screening of the capacity of a selection of these compounds (or their circulating metabolites) to modulate some risk factors for dismetabolic pathologies, iii) functional genomics of the anti-neoplastic properties of a selection of these compounds, and iv) identification of the ideal metabolic profile (phenotype) of the healthy apple.

The study will be carried out on healthy volunteers. A standardized cloudy juice will be used both plain and singularly fortified with 2 to 4 already demonstrated bioactive compounds (chosen among: quercetin 3-galactoside, ursolic acid, oligomeric procyanidins and chlorogenic acid), and a commercial pasteurized juice will be assumed as the reference blank. The cloudy juice will be processed under special conditions in order to have a composition equivalent to the fresh apple. Four compounds will be used for the in vitro and pilot in vivo study (a), and two compounds for the in vivo study (d).

The study will be organized as follows: a) in vivo pilot study of acute fresh standardized (fortified or not) apple juice consumption, against the blank juice, by blood sampling aimed at deriving a metabolomic profile, with the identification of the main metabolites present and their concentration, as well as the absorption kinetics of some of them, b) ex vivo study, to highlight the effect of the consumption of apple juice (fortified or not) on platelet aggregation and on the modulation of the transcriptome in leukocytes, done by blood sampling and addition of the derived serum of the volunteers to cell cultures; c) in vitro study on human endothelial cells, to evaluate the molecular effect of apple metabolites on different inflammation processes, done as in (b), d) in vivo study of chronic fresh apple juice consumption (crossover/double-blind), by blood sampling in order to determine (1) the influence of (fortified and not) apple juice consumption on biomarkers of degenerative pathologies (lipidemia, plasma and LDL redox state, platelet aggregation, inflammation markers), (2) the detailed metabolomic profile, including both the search of new biomarkers (untarget approach), as well as the plasma concentration of the apple compounds and metabolites identified in the pilot study (a), e) in vivo study of the anticancer power of the four bioactive compounds in the fortified juice in models of cancer stem cells of four solid tumors, done as in (b) and including the measure of cell kinetic parameters and indexes of tumor aggressiveness (viability, apoptosis, proliferation, clonogenicity, invasion), f) in vitro identification of the genomic determinants of the activity of the bioactive compound which will result endowed with the major anticancer power performed by a combination of transcriptome and “traslatome” profiling (changes in translating mRNAs), shRNA-induced gene silencing and bioinformatic data mining, g) in vivo study of the activity of this compound on xenotransplanted tumors grown in nude mice, and of its metabolites released in these mice.


WP5b Allergens

Besides the beneficial effects due to their consumption, apples are amongst the fruits that most frequently give rise to allergic reactions in an ever increasing number of consumers, both adults and children. A series of proteins with high allergenic properties and their associated genes have been identified by national and international research programmes previously established by groups involved in the present project. This basic knowledge has allowed to assess the overall allergenic potential and identify several important genetic and environmental factors governing it in the pre- and post-harvest phases, such as genotype, fruit load, storage, and elevation. Within the present project, different research lines will be pursued both by means of existing molecular tools and by developing new improved methods. The main initiatives will include: i) assessement of global fruit allergenicity exploiting apple genome and fruit transcriptomic profile;.ii) improvement and/or development of extraction methods optimized for apple fruit allergens; iii) development of specific antibodies to assay apple allergen content; using sera of allergic patients for testing the in vitro allergenic potential of apple samples; iv) assessing the allergenic potential of fruit produced by apple genotypes with pyramidised resistances; v) searching for hypoallergenic apple genotypes among those available in germplasm collections. In detail:

i) Clinical trials will be carried out in order to identify apple genotypes with divergent global fruit allergenic potential whose transcriptomic profiles will be assessed by means of high throughput technologies in order to establish putative association between the expression of specific gene sets and fruit allergenicity. This approach will allow to identify the master determinants of allergenicity to be further investigated at the genomic level.

ii) Currently used protein extraction methods could not be sufficiently specific and equally efficient for all the allergen classes known in apple, mainly because of the different extractability properties of allergenic proteins. Therefore the assessment of the allergenic potential of a protein extract could be strongly biased by the extraction technique, and the use of improved/new dedicated protocols with high efficiency and quality of the extract becomes crucial. Particular attention will be paid within the present project to the setting up and/or improvement of extraction techniques specific for apple fruit tissues (both cortex and epidermis). Different buffers will be tested and specific adjustments will be performed on available protocols in order to improve the extractability of the different allergen classes.

iii) The availability of specific antibodies is crucial for assessing the allergen content in protein extracts or whole fruit sections. Within the present project, specific antibodies will be developed for the most relevant allergens. For each allergen class the possibility of obtaining specific antibodies for the most allergenic isoforms will be exploited based upon preliminary bioinformatic studies carried out to characterize their antigenic properties and epitopes. Sera of allergic patients will be collected by allergologists already collaborating with the research groups herein involved, to be used both in the present project for assessing the overall allergenic profile of apple samples and in future initiatives.

iv) The allergenic potential of fruit produced by new genotypes with pyramidised resistance released by WP2 will be assessed by means of the available tools (i.e. qPCR, antibodies and sera). This evaluation is particularly important since most fruit allergens belong to the pathogenesis-related proteins (PR), whose expression might be modified in the genotypes with pyramidised resistance traits. The final goal of this research line will concern the identification of apple genotypes with an increased and durable resistance to some pathogens and a low allergen content. Nevertheless, an extensive screening will also be performed, by using the same analytical tools, on apple germplasm collections to search for hypoallergenic accessions. Finally, the allergenic properties of some of these fruits will be validated in vivo by skin tests carried out in collaboration with allergologists. The best genotypes will be used within point i) of the present WP and in future breeding programs.