tIn this study we aimed to identify genes that are responsive to pertussis toxin (PTx) and might eventu-ally be used as biological markers in a testing strategy to detect residual PTx in vaccines. By microarrayanalysis we screened six human cell types (bronchial epithelial cell line BEAS-2B, fetal lung fibroblastcell line MRC-5, primary cardiac microvascular endothelial cells, primary pulmonary artery smooth mus-cle cells, hybrid cell line EA.Hy926 of umbilical vein endothelial cells and epithelial cell line A549 andimmature monocyte-derived dendritic cells) for differential gene expression induced by PTx. Imma-ture monocyte-derived dendritic cells (iMoDCs) were the only cells in which PTx induced significantdifferential expression of genes. Results were confirmed using different donors and further extendedby showing specificity for PTx in comparison to Escherichia coli lipopolysaccharide (LPS) and Bordetellapertussis lipo-oligosaccharide (LOS). Statistical analysis indicated 6 genes, namely IFNG, IL2, XCL1, CD69,CSF2 and CXCL10, as significantly upregulated by PTx which was also demonstrated at the protein levelfor genes encoding secreted proteins. IL-2 and IFN- gave the strongest response. The minimal PTx con-centrations that induced production of IL-2 and IFN- in iMoDCs were 12.5 and 25 IU/ml, respectively.High concentrations of LPS slightly induced IFN- but not IL-2, while LOS and detoxified pertussis toxindid not induce production of either cytokine. In conclusion, using microarray analysis we evaluated sixhuman cell lines/types for their responsiveness to PTx and found 6 PTx-responsive genes in iMoDCs ofwhich IL2 is the most promising candidate to be used as a biomarker for the detection of residual PTx.
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From teh UU repository: "Background: Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, there are some concerns regarding its safety and long-term efficacy. The use of non-digestible oligosaccharides might improve OIT efficacy since they are known to directly modulate intestinal epithelial and immune cells in addition to acting as prebiotics. Aim: To investigate whether a diet supplemented with plant-derived fructo-oligosaccharides (FOS) supports the efficacy of OIT in a murine cow's milk allergy model and to elucidate the potential mechanisms involved. Methods: After oral sensitization to the cow's milk protein whey, female C3H/HeOuJ mice were fed either a control diet or a diet supplemented with FOS (1% w/w) and received OIT (10 mg whey) 5 days a week for 3 weeks by gavage. Intradermal (i.d.) and intragastric (i.g.) challenges were performed to measure acute allergic symptoms and mast cell degranulation. Blood and organs were collected to measure antibody levels and T cell and dendritic cell populations. Spleen-derived T cell fractions (whole spleen-and CD25-depleted) were transferred to naive recipient mice to confirm the involvement of regulatory T cells (Tregs) in allergy protection induced by OIT + FOS. Results: OIT + FOS decreased acute allergic symptoms and mast cell degranulation upon challenge and prevented the challenge-induced increase in whey-specific IgE as observed in sensitized mice. Early induction of Tregs in the mesenteric lymph nodes (MLN) of OIT + FOS mice coincided with reduced T cell responsiveness in splenocyte cultures. CD25 depletion in OIT + FOS-derived splenocyte suspensions prior to transfer abolished protection against signs of anaphylaxis in recipients. OIT + FOS increased serum galectin-9 levels. No differences in short-chain fatty acid (SCFA) levels in the cecum were observed between the treatment groups. Concisely, FOS supplementation significantly improved OIT in the acute allergic skin response, %Foxp3+ Tregs and %LAP+ Th3 cells in MLN, and serum galectin-9 levels. Conclusion: FOS supplementation improved the efficacy of OIT in cow's milk allergic mice. Increased levels of Tregs in the MLN and abolished protection against signs of anaphylaxis upon transfer of CD25-depleted cell fractions, suggest a role for Foxp3+ Tregs in the protective effect of OIT + FOS. "
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IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNβ1 and subsequently IL7. IFNβ1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors (e.g. ACE2, TMPRSS2, DPP4, CD46 and TNFRSF14), increases the expression of anti-viral proteins (e.g. RSAD2, AOS, ISG20 and Mx1) and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses.
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From PLoS website: In general, dietary antigens are tolerated by the gut associated immune system. Impairment of this so-called oral tolerance is a serious health risk. We have previously shown that activation of the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) by the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects both oral tolerance and food allergy. In this study, we determine whether a common plant-derived, dietary AhR-ligand modulates oral tolerance as well. We therefore fed mice with indole-3-carbinole (I3C), an AhR ligand that is abundant in cruciferous plants. We show that several I3C metabolites were detectable in the serum after feeding, including the high-affinity ligand 3,3´-diindolylmethane (DIM). I3C feeding robustly induced the AhR-target gene CYP4501A1 in the intestine; I3C feeding also induced the aldh1 gene, whose product catalyzes the formation of retinoic acid (RA), an inducer of regulatory T cells. We then measured parameters indicating oral tolerance and severity of peanut-induced food allergy. In contrast to the tolerance-breaking effect of TCDD, feeding mice with chow containing 2 g/kg I3C lowered the serum anti-ovalbumin IgG1 response in an experimental oral tolerance protocol. Moreover, I3C feeding attenuated symptoms of peanut allergy. In conclusion, the dietary compound I3C can positively influence a vital immune function of the gut.
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From the publisher: "Background: The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP). Main body: The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential. Conclusion: The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs." Authors: Jolanda H. M. van BilsenEmail author, Edyta Sienkiewicz-Szłapka, Daniel Lozano-Ojalvo, Linette E. M. Willemsen, Celia M. Antunes, Elena Molina, Joost J. Smit, Barbara Wróblewska, Harry J. Wichers, Edward F. Knol, Gregory S. Ladics, Raymond H. H. Pieters, Sandra Denery-Papini, Yvonne M. Vissers, Simona L. Bavaro, Colette Larré, Kitty C. M. Verhoeckx and Erwin L. Roggen
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From the article: "Scope: During food processing, the Maillard reaction ( М R) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β -lactoglobulin (BLG), in their interactions with cells crucially involved in allergy. Methods and results: BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T-cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco-2 monolayer. Uptake of glycated BLG by bone marrow–derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor-mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4 + T-cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG-specific IgE sensitized basophil cells. Conclusions: This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy."
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Abstract 1 Scope A major downside of oral immunotherapy (OIT) for food allergy is the risk of severe side effects. Non‐digestible short‐ and long‐chain fructo‐oligosaccharides (scFOS/lcFOS) reduce allergy development in murine models. Therefore, it is hypothesized that scFOS/lcFOS can also support the efficacy of OIT in a peanut allergy model. 2 Methods and Results After sensitization to peanut extract (PE) using cholera toxin, C3H/HeOuJ mice are fed a 1% scFOS/lcFOS or control diet and receive OIT (1.5 or 15 mg PE). Hereafter, mice are exposed to PE via different routes to determine the safety and efficacy of treatment in clinical outcomes, PE‐specific antibody production, and numbers of various immune cells. scFOS/lcFOS increases short‐chain fatty acid levels in the caecum and reduce the acute allergic skin response and drop in body temperature after PE exposure. Interestingly, 15 mg and 1.5 mg OIT with scFOS/lcFOS induce protection against anaphylaxis, whereas 1.5 mg OIT alone does not. OIT, with or without scFOS/lcFOS, induces PE‐specific immunoglobulin (Ig) IgG and IgA levels and increases CD103+ dendritic cells in the mesenteric lymph nodes. 3 Conclusions scFOS/lcFOS and scFOS/lcFOS combined with low dose OIT are able to protect against a peanut‐allergic anaphylactic response.
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This article gives information on an international ring trial of the epidermal-equivalent (EE) sensitizer potency assay.
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Despite changing attitudes towards animal testing and current legislation to protect experimental animals, the rate of animal experiments seems to have changed little in recent years. On May 15–16, 2013, the In Vitro Testing Industrial Platform (IVTIP) held an open meeting to discuss the state of the art in alternative methods, how companies have, can, and will need to adapt and what drives and hinders regulatory acceptance and use. Several key messages arose from the meeting. First, industry and regulatory bodies should not wait for complete suites of alternative tests to become available, but should begin working with methods available right now (e.g., mining of existing animal data to direct future studies, implementation of alternative tests wherever scientifically valid rather than continuing to rely on animal tests) in non-animal and animal integrated strategies to reduce the numbers of animals tested. Sharing of information (communication), harmonization and standardization (coordination), commitment and collaboration are all required to improve the quality and speed of validation, acceptance, and implementation of tests. Finally, we consider how alternative methods can be used in research and development before formal implementation in regulations. Here we present the conclusions on what can be done already and suggest some solutions and strategies for the future.
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