Functional differences between primary monocyte-derived and THP-1 macrophages and their response to LCPUFAs

Background: In immune cell models, macrophages are one of the most frequently used cell types. THP-1 cells are often used as model to study macrophage function, however they may act differently from primary human monocyte derived macrophages (MDMs). Methods: In this study, we investigated the intrinsic baseline differences between the human macrophage cell line THP-1 and human primary MDMs. Additionally, we studied the difference in response to treatment with long-chain polyunsaturated fatty acids (LCPUFAs): well-described immunomodulators. Results: Although the amount of cells that phagocytose were similar between the cell types, primary MDMs consumed significantly more E. coli bioparticles compared to THP-1 macrophages. In M1 macrophages, IL-12 secretion was almost fifty times higher by primary MDMs compared to THP-1 macrophages, thereby increasing the IL-12/IL-10 ratio. Despite this, the IL-12 secretion by THP-1 M1 macrophages was higher that the secretion of IL-10, thereby showing that it is still a suitable M1 type. Cytokine profiles differed between primary MDMs and THP-1 M1 and M2 macrophages. In response to LCPUFAs, primary M1 MDMs and THP-1 M1 macrophages were alike. Interestingly, primary M2 MDMs secreted less IL-10 and CCL22 when treated with LCPUFAs, whereas THP-1 M2 macrophages secreted more IL-10 when treated with LCPUFAs and showed no difference in CCL22 secretion. Conclusions: In conclusion, in an M1 setting, both THP-1 and primary MDMs are suitable models. However, when interested in M2 models, the model choice highly depends on the research question

IgE cross-reactivity measurement of cashew nut, hazelnut and peanut using a novel IMMULITE inhibition method

Tree nut-allergic individuals are often sensitised towards multiple nuts and seeds. The underlying cause behind a multi-sensitisation for cashew nut, hazelnut, peanut and birch pollen is not always clear. We investigated whether immunoglobulin E antibody (IgE) cross-reactivity between cashew nut, hazelnut and peanut proteins exists in children who are multi-allergic to these foods using a novel IMMULITE®-based inhibition methodology, and investigated which allergens might be responsible. In addition, we explored if an allergy to birch pollen might play a role in this co-sensitisation for cashew nut, hazelnut and peanut. Serum of five children with a confirmed cashew nut allergy and suffering from allergic symptoms after eating peanut and hazelnut were subjected to

Identification and in silico bioinformatics analysis of PR10 proteins in cashew nut

Proteins from cashew nut can elicit mild to severe allergic reactions. Three allergenic proteins have already been identified, and it is expected that additional allergens are present in cashew nut. pathogenesis-related protein 10 (PR10) allergens from pollen have been found to elicit similar allergic reactions as those from nuts and seeds. Therefore, we investigated the presence of PR10 genes in cashew nut. Using RNA-seq analysis, we were able to identify several PR10-like transcripts in cashew nut and cl

IgE Cross-Reactivity of Cashew Nut Allergens

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blot technique. Allergen cross-reactivity patterns between Anacardiaceae species were subsequently examined by SDS-PAGE and immunoblot inhibition, and IgE-reactive allergens were identified by LC-MS/MS. Results: From the 56 subjects analysed, 36 were positive on dot blot for cashew nut (63%). Of these, 50% were mono-sensitised to cashew nuts, 19% were co-sensitised to Anacardiaceae species, and 31% were co-sensitised to tree nuts. Subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. In pink peppercorn, putative albumin- and legumin-type seed storage proteins were found to cross-react with serum of cashew nut-sensitised subjects in vitro. In addition, a putative luminal binding protein was identified, which, among others, may be involved in cross-reactivity between several Anacardiaceae species. Conclusions: Results demonstrate the in vitro presence of IgE cross-sensitisation in children towards multiple Anacardiaceae species. In this study, putative novel allergens were identified in cashew, pistachio, and pink peppercorn, which may pose factors that underlie the observed cross-sensitivity to these species. The clinical relevance of this widespread cross-sensitisation is unknown.</p

IgE Cross-Reactivity of Cashew Nut Allergens

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blo

Cracking the cashew nut : Strategies to identify novel allergens

Cashew nut allergy has been recognized as a severe tree nut allergy amongst (Dutch) children and young adults and its prevalence seems to be increasing. For clinical diagnosis of an allergy, it is essential to know the causative agents in the food product causing the allergic symptoms.In this thesis entitled ‘Cracking the cashew nut: strategies to identify and characterize novel allergens’, we aimed to apply innovative strategies and technologies to identify and characterize putative allergenic proteins in cashew nut, to broaden the current knowledge on cashew nut allergens beyond those already known (Ana o 1, Ana o 2 and Ana o 3). Our knowledge of cashew nut proteins that can trigger an allergic reaction is currently very limited, especially compared to other nuts or seeds in which the allergen repertoire has been researched much more widely. Using several different strategies, we evidenced that additional allergens must be present in cashew nuts, which presumably contribute to the elicitation of allergic symptoms in cashew nut allergic patients. Knowledge of newly identified cashew nut proteins provides a basis for further research to extend clinical diagnostic tests and treatments currently available for cashew nut allergy.Chapter 2 includes an opinion on the use of current in vivo and ex vivo endpoints in murine food allergy models and their suitability for evaluating the sensitizing capacity of protein concentrates and/or food products. An overview is given of the best predictive risk assessment methods and endpoint parameters currently relied on in in vivo food allergy models with a focus on milk, egg and peanut allergens, addressing their strengths and limitations for assessing sensitization risks. Findings indicated that, although the current available models are suitable for studying the pathophysiology of food allergy, they still couldn’t predict the magnitude of the allergic potential of a particular allergen. Thus, there is still a strong need to better define the allergic reaction to predict the clinical outcomes of sensitization to novel food proteins. In addition, there is an urgent need for a consensus on key food allergy parameters to be applied in future food allergy research, to guarantee optimal lab- to-lab reproducibility and reliable use of predictive tests for protein risk assessment.Cashew nut allergic individuals may develop cross-reactive responses to foods that are phylogenetically related to cashew nut. In Chapter 3, we therefore aimed to determine the IgE cross-sensitisation and cross-reactivity profiles in cashew nut sensitised subjects. Profiling was specifically aimed at botanically related proteins of common tree nut species and other Anacardiaceae family members like pistachio, mango, pink peppercorn or sumac. Half of cashew nut positive sera on dot blot were co- sensitised; 19% to solely Anacardiaceae species and 31% to tree nuts, which indicated that cross-sensitisation/cross-reactivity is widespread among cashew nut allergic individuals. Interestingly, subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. Putative underlying novel allergens were identified in cashew nut, pistachio and pink peppercorn, which demonstrated that indeed additional allergens might exist in cashew nut that may pose factors underlying cashew nut allergic symptoms.In line with these findings, we applied a novel IMMULITE®-based inhibition methodology in Chapter 4, to investigate the IgE cross-reactivity between cashew nut-, hazelnut- and peanut proteins in children that are multi-allergic to these foods. Observations indicated that hazelnut extract was a strong inhibitor of cashew nut sIgE while cashew nut extract was less able to inhibit hazelnut extract. In contrast, peanut extract showed the least inhibition potency. Importantly, there were strong indications that a birch pollen sensitisation to Bet v 1 might play a role in the observed symptoms provoked upon ingestion of cashew nut and hazelnut, suggesting the existence of putative Bet v 1-like protein homologs in cashew nut.Based on the strong indications that additional allergenic proteins may exist in cashew nut, cashew nut transcript profiling was conducted resulting in a RNA-seq database that can be used to screen for protein homologs of allergens identified in phylogenic related species. In Chapter 5, we applied this method to identify and characterize three PR10 proteins in cashew nut. The identification and partial characterization of two additional 2S albumin proteins, next to the major cashew nut 2S albumin Ana o 3.0101, are described in Chapter 6.Finally, Chapter 7 discusses the major findings of the different research chapters and pros and cons of the applied strategies. Additional putative cashew nut allergens are presented, identified using the RNAseq screening approach mentioned in chapter 5 and 6 which, although not yet characterized, likely contribute to the allergen repertoire of cashew nut. To conclude, future research opportunities are presented that could take our current knowledge of cashew nut allergy to a higher level

Current Understanding of the Structure and Function of Fungal Immunomodulatory Proteins

Fungal immunomodulatory proteins (FIPs) are a group of proteins found in fungi, which are extensively studied for their immunomodulatory activity. Currently, more than 38 types of FIPs have been described. Based on their conserved structure and protein identity, FIPs can be classified into five subgroups: Fve-type FIPs (Pfam PF09259), Cerato-type FIPs (Pfam PF07249), PCP-like FIPs, TFP-like FIPs, and unclassified FIPs. Among the five subgroups, Fve-type FIPs are the most studied for their hemagglutinating, immunomodulating, and anti-cancer properties. In general, these small proteins consist of 110–125 amino acids, with a molecular weight of ~13 kDa. The other four subgroups are relatively less studied, but also show a noticeable influence on immune cells. In this review, we summarized the protein modifications, 3-dimensional structures and bioactivities of all types of FIPs. Moreover, structure-function relationship of FIPs has been discussed, including relationship between carbohydrate binding module and hemagglutination, correlation of oligomerization and cytokine induction, relevance of glycosylation and lymphocyte activation. This summary and discussion may help gain comprehensive understanding of FIPs' working mechanisms and scope future studies.</p

Influence of processing and in vitro digestion on the allergic cross-reactivity of three mealworm species

Edible insects are currently being evaluated as an alternative and more sustainable protein source for humans. The introduction of new food sources can lead to development of novel allergies. Because in the Western world, insects are unlikely to be consumed raw, it is important to know how processing and in vitro digestion might influence their allergenicity. Three edible mealworm species (Tenebrio molitor, Zophobas atratus and Alphitobius diaperinus) subjected to processing and in vitro digestion were analysed for IgE cross-reactivity. Immunoblot and MALDI-MS/MS analyses revealed that IgE from crustaceans or House dust mite (HDM) allergic patients showed cross-reactivity to mealworm tropomyosin or α-amylase, hexamerin 1B precursor and muscle myosin, respectively. Heat processing as well as in vitro digestion did diminish, but not eliminate, HDM or tropomyosin IgE cross-reactivity. Results show that individuals allergic to HDM or crustaceans might be at risk when consuming mealworms, even after heat processing.</p

Influence of processing and in vitro digestion on the allergic cross-reactivity of three mealworm species

Edible insects are currently being evaluated as an alternative and more sustainable protein source for humans. The introduction of new food sources can lead to development of novel allergies. Because in the Western world, insects are unlikely to be consumed raw, it is important to know how processing and in vitro digestion might influence their allergenicity. Three edible mealworm species (Tenebrio molitor, Zophobas atratus and Alphitobius diaperinus) subjected to processing and in vitro digestion were analysed for IgE cross-reactivity. Immunoblot and MALDI-MS/MS analyses revealed that IgE from crustaceans or House dust mite (HDM) allergic patients showed cross-reactivity to mealworm tropomyosin or α-amylase, hexamerin 1B precursor and muscle myosin, respectively. Heat processing as well as in vitro digestion did diminish, but not eliminate, HDM or tropomyosin IgE cross-reactivity. Results show that individuals allergic to HDM or crustaceans might be at risk when consuming mealworms, even after heat processing.</p