326 research outputs found
Plasma anandamide and other N-acylethanolamines are correlated with their corresponding free fatty acid levels under both fasting and non-fasting conditions in women
N-acylethanolamines (NAEs), such as anandamide (AEA), are a group of endogenous lipids derived from a fatty acid linked to ethanolamine and have a wide range of biological activities, including regulation of metabolism and food intake. We hypothesized that i) NAE plasma levels are associated with levels of total free fatty acids (FFAs) and their precursor fatty acid in fasting and non-fasting conditions and ii) moderate alcohol consumption alters non-fasting NAE levels. In a fasting and non-fasting study we sampled blood for measurements of specific NAEs and FFAs. In the fasting study blood was drawn after an overnight fast in 22 postmenopausal women. In the non-fasting study blood was sampled before and frequently after a standardized lunch with beer or alcohol-free beer in 19 premenopausal women. Fasting AEA levels correlated with total FFAs (r = 0.84; p <0.001) and arachidonic acid levels (r = 0.42; p <0.05). Similar results were observed for other NAEs with both total FFAs and their corresponding fatty acid precursors. In addition, AEA (r = 0.66; p <0.01) and OEA levels (r = 0.49;
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
Current (Food) allergenic risk assessment: is it fit for novel foods? status quo and identification of gaps
Food allergies are recognized as a global health concern. In order to protect allergic consumers from severe symptoms, allergenic risk assessment for well-known foods and foods containing genetically modified ingredients is installed. However, population is steadily growing and there is a rising need to provide adequate protein-based foods, including novel sources, not yet used for human consumption. In this context safety issues such as a potential increased allergenic risk need to be assessed before marketing novel food sources. Therefore, the established allergenic risk assessment for genetically modified organisms needs to be re-evaluated for its applicability for risk assessment of novel food proteins. Two different scenarios of allergic sensitization have to be assessed. The first scenario is the presence of already known allergenic structures in novel foods. For this, a comparative assessment can be performed and the range of cross-reactivity can be explored, while in the second scenario allergic reactions are observed toward so far novel allergenic structures and no reference material is available. This review summarizes the current analytical methods for allergenic risk assessment, highlighting the strengths and limitations of each method and discussing the gaps in this assessment that need to be addressed in the near future.Austrian Science Fund [FWF SFB F4603]; Ministry of Education, Science and Technological Development of the Republic of Serbia [OI172024]; MINECO, Spain [AGL2014-59771-R]; PROMAR: Projetos Pilotos e a Transformacao de Embarcacoes de Pesca [31-03-05-FEP-0060]info:eu-repo/semantics/publishedVersio
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
Dairy food structures influence the rates of nutrient digestion through different in vitro gastric behaviour
The purpose of this study was to investigate in vitro the extent to which specific food structures alter gastric behaviour and could therefore impact on nutrient delivery and digestion in the small intestine. Results obtained from a specifically developed gastric digestion model, were compared to results from a previous human study on the same foods. The semi-dynamic model could simulate the main gastric dynamics including gradual acidification, lipolysis, proteolysis and emptying. Two dairy-based foods with the same caloric content but different structure were studied. The semi-solid meal comprised a mixture of cheese and yogurt and the liquid meal was an oil in water emulsion stabilised by milk proteins. Our findings showed similar gastric behaviour to that seen previously in vivo. Gastric behaviour was affected by the initial structure with creaming and sedimentation observed in the case of liquid and semi-solid samples, respectively. Lipid and protein digestion profiles showed clear differences in the amount of nutrients reaching the simulated small intestine and, consequently, the likely bioaccessibility after digestion. The semi-solid sample generated higher nutrient released into the small intestine at an early stage of digestion whereas nutrient accessibility from liquid sample was delayed due to the formation of a cream layer in the gastric phase. This shows the strong effect of the matrix on gastric behaviour, proteolysis and lipolysis, which explains the differences in physiological responses seen previously with these systems in terms of fullness and satiety
Ohmic Heating for the dairy industry: a potential technology to develop probiotic dairy foods in association with modifications of whey protein structure
The use of whey in dairy probiotics is a topic of great interest to the scientific community and the food industries. However, few studies address the effect of ohmic heating (OH) on cell metabolism and growth parameters of probiotic microorganisms. Despite of this, OH under sub-lethal conditions presents promising results regarding the enhancement of growth rate and bacteriocin activity, leading to considerable improvements in the fermentation process. Thus, this review highlights the main findings and advances on the effect of OH on probiotic metabolism, while addressing the modification of whey protein structure as potential carrier of probiotic entities, aiming at stimulating interest and encouraging the development of functional products using OH.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 — Programa Operacional Regional do Norte. Pedro Santos is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15), funded by the European Social Fund under the scope of Norte2020 — Programa Operacional Regional do Norte. Ricardo Pereira is recipient of a fellowship supported by FCT (SFRH/BPD/81887/2011).info:eu-repo/semantics/publishedVersio
Influence of ohmic heating on the structural and immunoreactive properties of soybean proteins
Ohmic heating (OH) encompasses interesting benefits towards thermal processing. Envisaging an increasing relevance of soybean protein as an alternative non-animal protein, it is important to understand how OH can contribute to the quality and immunoreactivity of soybean-derived products. This study describes, for the first time, the impact of OH when applied at different electrical frequencies (50 Hz20 kHz) and moderate electric field intensities (up to 20 V/cm), on the leakage of metals from the electrodes and immunoreactivity aspects of soybean protein isolate (SPI). This was achieved by monitoring the occurrence of electrochemical reactions and evaluating IgG-binding capacity. OH performed at 50 Hz and 95 °C induced significant alterations on the intrinsic fluorescence of SPI (p 0.05) and the release of detectable amounts of Fe/Ni, with a subsequent reduction of 36% in the immunoreactivity of Gly m TI. The occurrence of non-thermal effects, as well as the interaction between protein and trace metals, may result in a partial blockage of protein epitopes, thus impairing specific antibody binding. These findings present novel information about the importance of OH parameters, such as electrical frequency and occurrence of electrochemical reactions, which can affect the structure and immunoreactivity of SPI fractions.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 and UID/QUI/50006/2020 with funding from FCT/MCTES
through national funds, and AgriFood XXI R&D&I project, operation number NORTE-01-0145-FEDER-000041 and NORTE-01-0145-FEDER 000052, co-financed by the European Regional Development Fund
(FEDER) through NORTE 2020 (Northern Regional Operational Pro gram 2014/2020). This work also received financial support from the European Union (FEDER funds through COMPETE POCI-01-0145-
FEDER-031720) and National Funds (FCT) through project Alle Risk Assess PTDC/BAA-AGR/31720/2017. Caterina Villa and Luís Machado thank FCT for their grants under project AlleRiskAssess
(PTDC/BAA-AGR/31720/2017). Joana Costa thanks FCT for funding through program DL 57/2016 – Norma transitoria (SFRH/BPD/102404/2014). Ricardo N. Pereira acknowledge FCT for its Assistant Research
contract obtained under CEEC Individual 2017.info:eu-repo/semantics/publishedVersio
Inter-laboratory reproducibility of fast gas chromatography–electron impact–time of flight mass spectrometry (GC–EI–TOF/MS) based plant metabolomics
The application of gas chromatography–mass spectrometry (GC–MS) to the ‘global’ analysis of metabolites in complex samples (i.e. metabolomics) has now become routine. The generation of these data-rich profiles demands new strategies in data mining and standardisation of experimental and reporting aspects across laboratories. As part of the META-PHOR project’s (METAbolomics for Plants Health and OutReach: http://www.meta-phor.eu/) priorities towards robust technology development, a GC–MS ring experiment based upon three complex matrices (melon, broccoli and rice) was launched. All sample preparation, data processing, multivariate analyses and comparisons of major metabolite features followed standardised protocols, identical models of GC (Agilent 6890N) and TOF/MS (Leco Pegasus III) were also employed. In addition comprehensive GC×GC–TOF/MS was compared with 1 dimensional GC–TOF/MS. Comparisons of the paired data from the various laboratories were made with a single data processing and analysis method providing an unbiased assessment of analytical method variants and inter-laboratory reproducibility. A range of processing and statistical methods were also assessed with a single exemplary dataset revealing near equal performance between them. Further investigations of long-term reproducibility are required, though the future generation of global and valid metabolomics databases offers much promise
Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis
Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs
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