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The European Commission asked EFSA to revise the risk assessment for honey bees, bumble bees and solitary bees. This guidance document describes how to perform risk assessment for bees from plant protection products, in accordance with Regulation (EU) 1107/2009. It is a review of EFSA's existing guidance document, which was published in 2013. The guidance document outlines a tiered approach for exposure estimation in different scenarios and tiers. It includes hazard characterisation and provides risk assessment methodology covering dietary and contact exposure. The document also provides recommendations for higher tier studies, risk from metabolites and plant protection products as mixture., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Currently, there is a growing interest in developing biopesticides and increasing their share in the plant protection market as sustainable tools in integrated pest management (IPM). Therefore, it is important that regulatory requirements are consistent and thorough in consideration of biopesticides' unique properties. While microbial pesticides generally have a lower risk profile, they present special challenges in non-target organism testing and risk assessment since, in contrast to chemical pesticides, their modes of action include infectivity and pathogenicity rather than toxicity alone. For this reason, non-target organism testing guidelines designed for conventional chemical pesticides are not necessarily directly applicable to microbial pesticides. Many stakeholders have recognised the need for improvements in the guidance available for testing microbial pesticides with honey bees, particularly given the increasing interest in development and registration of microbial pesticides and concerns over risks to pollinators. This paper provides an overview of the challenges with testing and assessment of the effects of microbial pesticides on honey bees ( Apis mellifera ), which have served as a surrogate for both Apis and non- Apis bees, and provides a foundation toward developing improved testing methods., Competing Interests: Conflict of interest The authors declare no competing interests.

Background: Peanut, tree nut, and sesame allergies are responsible for most life-threatening food-induced allergic reactions. Rates of coexistent allergy between these foods have been from mostly retrospective studies that include only a limited number of tree nuts or were not based on oral food challenges., Objective: The Pronuts study is a multicenter European study (London, Geneva, and Valencia) assessing the challenge-proven rate of coexistent peanut, tree nut, and/or sesame seed allergy., Methods: Children aged 0 to 16 years with at least 1 confirmed nut or sesame seed allergy underwent sequential diagnostic food challenges to all other nuts and sesame seed., Results: Overall, the rate of coexistent peanut, tree nut, and sesame seed allergy was 60.7% (n = 74/122; 95% CI, 51.4% to 69.4%). Peanut allergy was more common in London, cashew and pistachio nut allergies were more common in Geneva, and walnut and pecan allergies were more common in Valencia. Strong correlations were found between cashew-pistachio, walnut-pecan, and walnut-pecan-hazelnut-macadamia clusters. Age (>36 months) and center (Valencia > Geneva > London) were associated with an increased odds of multiple nut allergies. By pursuing the diagnostic protocol to demonstrate tolerance to other nuts, participants were able to introduce a median of 9 nuts., Conclusion: We found a higher rate of coexistent nut and sesame seed allergies than previously reported. Performing sequential food challenges was labor intensive and could result in severe allergic reactions; however, it reduced dietary restrictions. Age was a significant predictor of multiple nut allergies, and thus the secondary spread of nut allergies occurred in older children., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

To date, only few studies have assessed oral immunotherapy (OIT) for wheat allergy and often describe severe adverse reactions during therapy. We developed partially hydrolyzed wheat-based cereals (pHC), which were used in a multicenter, open-label, OIT pilot study, in immunoglobulin E-mediated wheat allergy children (NCT01332084). The primary objective of the study was to test whether wheat allergic patients tolerate pHC and primary end point was the presence or not of immediate adverse reactions to pHC during the 1-day initial escalation phase (stepwise increased doses of pHC), with evaluation of the maximum dose tolerated. Of the 9 patients enrolled in the trial, 4 discontinued OIT because of mild to severe reactions at the initial escalation phase. The 5 patients who passed the escalation phase consumed pHC daily for 1 to 6 months. One of these patients withdrew due to noncompliance, whereas the 4 others completed the study and successfully passed the wheat challenge test at the end of the study. About 60% of the adverse events were unrelated to the study product. Our study provides preliminary evidence that pHC is tolerated by a subset of wheat allergic patients. Further studies are warranted to test its efficacy as a potential therapeutic option for wheat allergic patients., Competing Interests: Declaration of Conflicting Interests:The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: S.N., S.P., A.M., Y.M.V., M.F., R.F., A.W., and A.Z. are or were employees of Nestec Ltd.

Background: Data on patients affected by chronic mucocutaneous candidiasis underscore the preponderant role of IL-17 receptor A (IL-17RA) in preserving mucocutaneous immunity. Little is known about the role of adenosine deaminase (ADA) 2 in regulation of immune responses, although recent reports linked ADA2 deficiency with inflammation and vasculitis., Objective: We sought to investigate the mechanisms of chronic inflammation and vasculitis in a child lacking IL-17RA and ADA2 to identify therapeutic targets., Methods: We report a family with 2 siblings who have had recurrent mucocutaneous infections with Candida albicans and Staphylococcus aureus and chronic inflammatory disease and vasculitis since early childhood, which were refractory to classical treatments. Array-based comparative genomic hybridization analysis showed that both siblings are homozygous for a 770-kb deletion on chr22q11.1 encompassing both IL17RA and cat eye critical region 1 (CECR1). Immunologic studies were carried out by means of flow cytometry, ELISA, and RIA., Results: As expected, in the affected child we found a lack of IL-17RA expression, which implies a severe malfunction in the IL-17 signaling pathway, conferring susceptibility to recurrent mucocutaneous infections. Surprisingly, we detected an in vitro and in vivo upregulation of proinflammatory cytokines, notably IL-1β and TNF-α, which is consistent with the persistent systemic inflammation., Conclusions: This work emphasizes the utility of whole-genome analyses combined with immunologic investigation in patients with unresolved immunodeficiency. This approach is likely to provide an insight into immunologic pathways and mechanisms of disease. It also provides molecular evidence for more targeted therapies. In addition, our report further corroborates a potential role of ADA2 in modulating immunity and inflammation., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: There is promising but conflicting evidence to recommend the addition of probiotics to foods for prevention and treatment of allergy. Based on previous studies with fermented milk containing Lactobacillus paracasei NCC2461, we aimed to compare the effect of a powder form of the latter probiotic with the effect of a blend of Lactobacillus acidophilus ATCC SD5221 and Bifidobacterium lactis ATCC SD5219 in patients with allergic rhinitis., Methods: A double-blind, randomized, cross-over study, involving 31 adults with allergic rhinitis to grass pollen, was performed outside the grass pollen season (registration number: NCT01233154). Subjects received each product for 4-weeks in two phases separated by a wash-out period of 6 to 8 weeks. A nasal provocation test was performed before and after each 4-week product intake period, and outcome parameters (objective and subjective clinical symptoms; immune parameters) were measured during and/or 24 hours after the test., Results: Out of the 31 subject enrolled, 28 completed the study. While no effect was observed on nasal congestion (primary outcome), treatment with NCC2461 significantly decreased nasal pruritus (determined by VAS), and leukocytes in nasal fluid samples, enhanced IL-5, IL-13 and IL-10 production by peripheral blood mononuclear cells in an allergen specific manner and tended to decrease IL-5 secretion in nasal fluid, in contrast to treatment with the blend of L. acidophilus and B. lactis., Conclusions: Despite short-term consumption, NCC2461 was able to reduce subjective nasal pruritus while not affecting nasal congestion in adults suffering from grass pollen allergic rhinitis. The associated decrease in nasal fluid leukocytes and IL-5 secretion, and the enhanced IL-10 secretion in an allergen specific manner may partly explain the decrease in nasal pruritus. However, somewhat unexpected systemic immune changes were also noted. These data support the study of NCC2461 consumption in a seasonal clinical trial to further demonstrate its potentially beneficial effect.

Background: Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen., Methods: Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen., Results: The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing., Conclusions: The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human., Trial Registration: ClinicalTrials.gov NCT01719133.

Background: Children with atopic diseases in early life are frequently found with positive IgE tests to peanuts/tree nuts without a history of previous ingestion. We aimed to identify risk factors for reactions to nuts at first introduction., Methods: A retrospective case-note and database analysis was performed. Recruitment criteria were: patients aged 3-16 yr who had a standardized food challenge to peanut and/or tree nuts due to sensitisation to the peanut/tree nut (positive spIgE or SPT) without previous consumption. A detailed assessment was performed of factors relating to food challenge outcome with univariate and multivariate logistic regression analysis., Results: There were 98 food challenges (47 peanut, 51 tree nut) with 29 positive, 67 negative and 2 inconclusive outcomes. A positive maternal history of allergy and a specific IgE >5 kU/l were strongly associated with a significantly increased risk of a positive food challenge (OR 3.73; 95% CI 1.31-10.59; p = 0.013 and OR 3.35; 95% CI 1.23-9.11; p = 0.007, respectively). Adjusting for age, a three year-old with these criteria has a 67% probability of a positive challenge. There was no significant association between types of peanut/tree nut, other food allergies, atopic conditions or severity of previous food reactions and positive challenges., Conclusions: We have demonstrated an association between the presence of maternal atopic history and a specific IgE >5 kU/l, with a significant increase in the likelihood of a positive food challenge. Although requiring further prospective validation these easily identifiable components should be considered when deciding the need for a challenge., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Background: Food allergy in children significantly affects their quality of life. Its impact can be analyzed by quality of life questionnaires., Objectives: The aim of our study was to validate the French version of disease-specific questionnaires and to evaluate the quality of life in children with IgE-mediated food allergy., Methods: Two validated food allergy-specific questionnaires for quality of life, the parent's and children's forms (FAQLQ-PF and FAQLQ-CF), were translated from English to French and submitted to children with food allergy and their parents. Questionnaires were analyzed in terms of emotional impact, food anxiety, and social and food limitations. NCT 01480427., Results: Sixty-two parents of children aged 0-12 yrs answered the FAQLQ-PF, and 32 children aged 8-12 yrs the FAQLQ-CF. Construct validity of both questionnaires was assessed by correlation between the FAQLQs and FAIM (r = 0.85 and 0.84, respectively). Both FAQLQs had good internal consistency (Cronbach's α = 0.748 and 0.67, respectively). Young children (0-3 yrs old) showed better quality of life scores than older children (FAQLQ-PF global score: p = 0.02). Worse scores were also shown among children with previous severe systemic reactions (FAQLQ-PF global score: p = 0.039), the ones with an allergic mother (FAQLQ-PF global score: p = 0.002), or allergic siblings (FAQLQ-PF emotional impact score: p = 0.034), the ones with multiple food allergy (more than 1 food) (FAQLQ-PF anxiety score: p = 0.04) and among the girls (FAQLQ-CF global score: p = 0.031)., Conclusion: Older children, the ones with severe systemic reactions, or with mothers or siblings also affected by allergies, as well as girls, and children with multiple food allergies show worse quality of life scores., (© 2012 John Wiley & Sons A/S.)

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.

Objective: To compare the performance of prototype Access® sVEGF R1 and PlGF automated immunoassays from Beckman Coulter to the Quantikine® microplate ELISA assays by R&D Systems., Methods: Samples obtained from pregnant women, non-pregnant women and men were assayed according to manufacturers' instructions., Results: Compared to the Quantikine assays, the Access assays demonstrated improved precision, increased sensitivity, broader dynamic ranges, and reduced analysis time. The Access assays were found to be specific for free sVEGF R1 and free PlGF., Conclusion: There was good correlation between the Access and Quantikine assays. Superior performance by Access assays may have important prenatal diagnostic implications.

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket that also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. Although this domain is essential for Hsp90 function, the molecular basis for adenosine nucleotide-dependent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, remains to be established. We report that bis-ANS (1,1'-bis(4-anilino-5-napthalenesulfonic acid), an environment sensitive fluorophore known to interact with nucleotide-binding domains, binds to the adenosine nucleotide-binding domain of GRP94 and thereby activates its molecular chaperone and peptide binding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activates GRP94 function. bis-ANS activation of GRP94 function was efficiently blocked by radicicol, an established inhibitory ligand for the adenosine nucleotide binding pocket. Confirmation of the N-terminal nucleotide binding pocket as the bis-ANS-binding site was obtained following covalent incorporation of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labeled limit digestion products. These data identify a ligand dependent regulation of GRP94 function and suggest a model whereby GRP94 function is regulated through a ligand-dependent conversion of GRP94 from an inactive to an active conformation.

Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elicits cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell-derived peptides. Elicitation of a CTL response requires that GRP94-associated peptides be transferred onto major histocompatability complex (MHC) class I molecules, a process that is postulated to accompany GRP94 internalization by antigen presenting cells, such as macrophages (Mphi) and dendritic cells (DC). In studies of GRP94 uptake in elicited Mphi, we report that Mphi display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis. GRP94 internalized by this pathway co-localized predominately with transferrin-positive early endosomes. At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed. When GRP94 was present in the medium, and thus accessible to both receptor-mediated and fluid phase internalization pathways, internalization was modestly inhibited in the presence of yeast mannan, a competitive inhibitor of mannose/fucose receptor activity, and substantially inhibited by dimethylamiloride, an inhibitor of macropinocytosis. GRP94 internalized via macropinocytosis did not display prominent co-staining with the lysosomal marker LAMP-2. These data identify multiple pathways of GRP94 internalization and indicate that receptor-dependent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety. Most significantly, these data demonstrate the existence of cell surface receptor(s), apparently unique to antigen presenting cells, that function in the binding and internalization of the ER chaperone GRP94.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.

Dopamine and its conjugates are widely distributed among biological species and are utilized for a variety of functions. Insects metabolize dopamine for cuticle melanization and sclerotization. Among the most abundant dopamines found in the larval and pupal development stages of Manduca sexta, the tobacco hornworm, are N-acetyldopamine and N-beta-alanyldopamine. In addition, glycosylated derivatives of these dopamines are found mainly in the hemolymph just prior to cuticulogenesis. The 1H and 13C NMR resonances of dopamine, its 3-O-methyl, 4-O-methyl, N-acetyl, and N-beta-alanyl derivatives, norepinephrine, 4-O-(beta-D-glucuronopyranosyl)dopamine, and the glycosylated products of N-beta-alanyldopamine and dopamine have largely been assigned. Assignments were based on one- and two-dimensional NMR analyses of the above compounds combined with that of specifically enriched [C7-13C]dopamine. 1H NMR showed that the major glycosylated natural product isolated from M. sexta pupal hemolymph was a 3-O-glycosyl derivative of N-beta-alanyldopamine. 13C NMR confirmed that the carbohydrate was D-glucose probably in a beta-linkage. 1H NMR of the aromatic ring protons provided the most definitive method to distinguish 3-O- from 4-O-derivatives of dopamine. In addition, the 3-O-glucosyl conjugate of N-beta-alanyldopamine had unique chemical shifts and coupling patterns compared to those of 4-O-(beta-D-glucuronosyl)- and 3-O-(beta-D-glucopyranosyl)dopamine.