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Background The pathogenesis of chronic spontaneous urticaria (CSU), including the mechanism of action of omalizumab, remain unclear. We hypothesized complement system involvement given the often fast clinical response induced by treatment, including omalizumab. Therefore, we assessed the role of various complement factors surrounding omalizumab treatment. Methods Thirty CSU patients (median age 42 [range 21–70]; 73 % female) with a median once daily Urticaria Activity Score over 7 days (UAS7) score at baseline of 31.5 points were enrolled. Treatment consisted of six administrations of 300 mg omalizumab every 4 weeks succeeded by a follow‐up period of 12 weeks. Four punch skin biopsies were taken per patient; at baseline from lesional skin, at baseline from nonlesional skin, and after 1 and 7 days from formerly lesional skin. Complement activity, including C1q, C3, C3bc/C3, C4, C4bc/C4, C5a, and Membrane Attack Complex in peripheral blood were analyzed and complement activation in the skin was determined by the analysis of C4d deposition. Results were related to the clinical response to omalizumab. Results Fifteen patients showed a UAS7 score of 6 or lower (median 0) at Week 24, 15 patients did not (median 16). Lesional skin biopsies at baseline revealed complement deposition (C4d) in blood vessels in the papillary dermis of 53% (16/30) of the patients, which suggests involvement of immune complexes in the pathogenesis of urticaria. Moreover, indication of increased complement activation in CSU was substantiated by increased C5a levels in peripheral blood compared to healthy controls (p = 0.010). The clinical effect of omalizumab could not be linked to the variation of complement components. Conclusions Both C4d deposition in lesional skin and elevated C5a levels in peripheral blood indicate the involvement of complement activation in the pathogenesis of CSU. No correlation was found between omalizumab and activation of complement indicative of independent processes in the immunopathogenesis of CSU.

Background Data on the impact of the number and nature of perceived asthma triggers on health‐related quality of life (HRQL) in children are scarce. Objective To investigate the impact of perceived asthma triggers on both asthma‐specific and generic HRQL in children. Methods A cross‐sectional study was conducted among children (7‐18 years) with asthma in secondary and tertiary care. Children were screened with electronic questionnaires regarding respiratory and allergic symptoms. Asthma‐specific HRQL was assessed using the Pediatric Asthma Quality of Life Questionnaire (PAQLQ) (score range 1‐7) and generic HRQL using the RAND questionnaire (score range 7‐32). The Kruskal‐Wallis test and one‐way ANOVA were used to test the difference of, respectively, the PAQLQ and RAND scores across the number of perceived asthma triggers (0, 1‐2, 3‐4, or ≥ 5). Univariable and multivariable linear regression analyses were performed to evaluate the association between individual triggers and HRQL. Results A total of 527 children with a mean (SD) age of 12.1 (2.9) years were included. Children with a higher number of perceived triggers had significantly lower PAQLQ and RAND scores (ie poorer HRQL). The difference in PAQLQ scores was clinically relevant between children with 0 versus 3‐4 or ≥ 5 triggers and 1‐2 versus ≥ 5 triggers (mean difference 0.66, 1.02 and 0.63, respectively). Especially, non‐allergic triggers (physical exercise, the weather, (cigarette) smoke and emotions) were significantly associated with reduced PAQLQ scores. Emotions and food/drinks were associated with reduced RAND scores. Conclusion and Clinical Relevance A higher number of perceived triggers of asthma were associated with reduced HRQL in children with asthma. Especially, non‐allergic triggers were associated with reduced HRQL.

Human monoclonal antibodies (mAbs) are valuable tools to link genetic information with functional features and to provide a platform for conformational epitope mapping. Additionally, combined data on genetic and functional features provide a valuable mosaic for systems immunology approaches. Strategies to generate human mAbs from peripheral blood have been described and used in several studies including single cell sequencing of antigen-binding B cells and the establishment of antigen-specific monoclonal Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCLs). However, direct comparisons of these two strategies are scarce. Hence, we sought to set up these two strategies in our laboratory using peanut 2S albumins (allergens) and the autoantigen anti-Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2, alternatively ‘ARHGDIB’) as antigen targets to directly compare these strategies regarding costs, time expenditure, recovery, throughput and complexity. Regarding single cell sequencing, up to 50% of corresponding V(D)J gene transcripts were successfully amplified of which 54% were successfully cloned into expression vectors used for heterologous expression. Seventy-five percent of heterologously expressed mAbs showed specific binding to peanut 2S albumins resulting in an overall recovery of 20.3%, which may be increased to around 29% by ordering gene sequences commercially for antibody cloning. In comparison, the establishment of monoclonal EBV-LCLs showed a lower overall recovery of around 17.6%. Heterologous expression of a mAb carrying the same variable region as its native counterpart showed comparable concentration-dependent binding abilities. By directly comparing those two strategies, single cell sequencing allows a broad examination of antigen-binding mAbs in a moderate-throughput manner, while the establishment of monoclonal EBV-LCLs is a powerful tool to select a small number of highly reactive mAbs restricted to certain B cell subpopulations. Overall, both strategies, initially set-up for peanut 2S albumins, are suitable to obtain human mAbs and they are easily transferrable to other target antigens as shown for ARHGDIB.

Background Component-resolved diagnostics (CRD) help predict hazelnut allergy (HA) in children, but are of unknown diagnostic value in adults. This study aimed to evaluate the diagnostic accuracy of IgE to hazelnut extract and components in adults. Methods A Dutch population of consecutively presenting adults suspected of HA, who underwent a double-blind placebo-controlled food challenge, were included. Serum IgE to hazelnut extract and Cor a 1, 8, 9 and 14 was measured on ImmunoCAP. Diagnostic accuracy was assessed by area under the curve (AUC) analysis. Results Of 89 patients undergoing challenge, 46 had challenge-confirmed HA: 17 based on objective and 29 based on subjective symptoms. At commonly applied cutoffs 0.1 and 0.35 kUA /L, high sensitivity was observed for IgE to hazelnut extract and Cor a 1 (range 85-91%), and high specificity for IgE to Cor a 8, 9 and 14 (range 77-95%). However, the AUCs for hazelnut extract and components were too low for accurate prediction of HA (range 0.50-0.56). Combining hazelnut extract and component IgE measurements did not significantly improve accuracy. Higher IgE levels to Cor a 9 and 14 were tentatively associated with HA with objective symptoms, but the corresponding AUCs still only reached 0.68 and 0.63 respectively. Conclusions Although hazelnut allergic adults are generally sensitized to hazelnut extract and Cor a 1, and hazelnut tolerant adults are usually not sensitized to Cor a 8, 9 or 14, challenge testing is still needed to accurately discriminate between presence and absence of HA in adults from a birch-endemic country.

Background Specific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin‐specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors. Methods 2S albumin‐binding B‐cells were single‐cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non‐atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering. Results The frequency of 2S albumin‐binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non‐atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non‐specific mAbs (9/10) were mainly derived from non‐atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non‐atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances. Conclusions Peanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs)., 2S albumin‐specific IgM+B cells from peanut allergic patients show partly a high number of non‐silent mutations. VH3‐family genes are predominantly used in 2S albumin‐specific B‐cells of peanut allergic patients. Certain public antibody sequences (HCDR3 sequence motifs) are associated with peanut allergy or tolerance in patients sensitized to Ara h 2 and/or 6. Abbreviations: BCR, B cell receptor; HCDR3, heavy chain complementarity‐determining region 3; VH, variable (V) gene of the heavy chain

Background Allergen information on product labels is crucial in food allergy management, though inadequacy in current labelling practices is one of the major causes for accidental reactions upon consuming prepacked food products. Objective This study analyses current status of communicating allergen information on food labels and provides practical recommendations for improving the label format based on communication theory. Methods Product labels (N 288) of seven food categories from private label products and brands were obtained at three retailers in the Netherlands. Information regarding the 14 EU‐regulated allergens was evaluated by the frequency of emphasizing allergens in the ingredient list, use of precautionary allergen labelling (PAL), icons and an allergen information section. Effectiveness of communication was assessed evaluating readability and findability of information on allergens using principles of Gestalt and Cognitive Load theories. Results As requested by EU regulation 1169/2011, emphasizing allergens in the ingredient list was almost 100%, all other presentations of information on allergens on labels was highly diverse. A separate allergen information section was present on most private label products. This section could, but not necessarily did, repeat allergens from the ingredient list and/or give a PAL. Brands often provided a PAL at the end of the ingredient list. Part of the products displayed an icon at different locations of the label. Label background, a lack of cohesion and variation in location of topics hamper the identification of relevant information on allergens by (allergic) consumers. Recommendations include a standardized order for mandatory and voluntary topics on the label and a separate allergen information section. Conclusion and clinical relevance Overall, consumers encounter a wide and inconsistent range in ways of presentation of allergen information on labels. Standardization according to basic design principles can improve usability and support safe food purchases for allergic consumers.

Background:It is not well-understood why symptom severity varies between patients with peanut allergy (PA).Objective:To gain insight into the clinical profile of subjects with mild-to-moderate and severe PA, and investigate individual and collective predictive accuracy of clinical background and IgE to peanut extract and components for PA severity.Methods:Data on demographics, patient history and sensitization at extract and component level of 393 patients with probable PA (symptoms ≤ 2 h + IgE sensitization) from 12 EuroPrevall centers were analyzed. Univariable and penalized multivariable regression analyses were used to evaluate risk factors and biomarkers for severity.Results:Female sex, age at onset of PA, symptoms elicited by skin contact with peanut, family atopy, atopic dermatitis, house dust mite and latex allergy were independently associated with severe PA; birch pollen allergy with mild-to-moderate PA. The cross-validated AUC of all clinical background determinants combined (0.74) was significantly larger than the AUC of tests for sensitization to extract (0.63) or peanut components (0.54–0.64). Although larger skin prick test wheal size, and higher IgE to peanut extract, Ara h 1 and Ara h 2/6, were associated with severe PA, and higher IgE to Ara h 8 with mild-to-moderate PA, addition of these measurements of sensitization to the clinical background model did not significantly improve the AUC.Conclusions:Models combining clinical characteristics and IgE sensitization patterns can help establish the risk of severe reactions for peanut allergic patients, but clinical background determinants are most valuable for predicting severity of probable PA in an individual patient.

BackgroundDendritic cells (DCs) play an important role in antigen presentation, and are an interesting target for immune-modulation in allergies. Short- and long-chain fructo-oligosaccharides (scFOS/lcFOS, FF) have immunomodulatory capacities, and may influence the outcome of DC antigen presentation.ObjectiveThis study investigated the effect of FF during DC maturation and allergen presentation using cells of peanut-allergic patients in an autologous DC-T cell assay.MethodsCD14+and CD4+T cells were isolated from peanut-allergic patients. CD14+monocytes were differentiated into immature DCs (imDCs), and matured (matDCs) in the presence or absence of crude peanut-extract (CPE) and/or FF, and co-cultured in an autologous DC-T cell assay. T cell polarization, proliferation and cytokine production were measured.ResultsExpression of maturation surface molecule markers on matDCs was not affected by CPE and/or FF. By contrast, the IL-10 secretion by matDCs increased compared to imDCs, upon exposure to CPE and FF compared to CPE alone. Also the IP-10 secretion increased in CPE/FF-matDCs compared to imDC. CPE-matDCs enhanced IL-13 release in the DC-T-cell assay and Treg polarization in presence or absence of FF. CPE/FF-DCs tended to increase the Treg/Th1 and Treg/Th2 ratios compared to matDCs. The proliferation of both Treg and Th2 cells tended to increase when T cells were co-cultured with CPE-matDCs compared to matDCs, which became significant when CPE-matDCs were also exposed to FF and a same tendency was shown for Th1 proliferation.ConclusionOnly in the presence of FF, CPE-matDCs produced increased regulatory and Th1-related mediators. CPE-matDCs modified T cell polarization and proliferation, and additional exposure to FF tended to enhance Treg/Th2 and Treg/Th1 ratios instructed by CPE/FF-matDCs. However this effect was not strong enough to suppress CPE-matDCs induced IL-13 release by Th-cells. This indicates the ability of FF to modify DC maturation in the presence of an allergen supporting a more Treg/Th1 prone direction of the successive allergen specific Th2 cell response.

Lupine flour is a valuable food due to its favorable nutritional properties. In spite of its allergenic potential, its use is increasing. Three lupine species, Lupinus angustifolius, L. luteus, and L. albus are relevant for human nutrition. The aim of this study is to clarify whether the species differ with regard to their allergen composition and whether anaphylaxis marker allergens could be identified in lupine. Patients with the following characteristics were included: lupine allergy, suspected lupine allergy, lupine sensitization only, and peanut allergy. Lupine sensitization was detected via CAP-FEIA (ImmunoCAP) and skin prick test. Protein, DNA and expressed sequence tag (EST) databases were queried for lupine proteins homologous to already known legume allergens. Different extraction methods applied on seeds from all species were examined by SDS-PAGE and screened by immunoblotting for IgE-binding proteins. The extracts underwent different and successive chromatography methods. Low-molecular-weight components were purified and investigated for IgE-reactivity. Proteomics revealed a molecular diversity of the three species, which was confirmed when investigated for IgE-reactivity. Three new allergens, L. albus profilin, L. angustifolius and L. luteus lipid transfer protein (LTP), were identified. LTP as a potential marker allergen for severity is a valuable additional candidate for molecular allergy diagnostic tests.

Background: The heterogeneity and lack of validation of existing severity scores for food allergic reactions limit standardization of case management and research advances. We aimed to develop and validate a severity score for food allergic reactions. Methods: Following a multidisciplinary experts consensus, it was decided to develop a food allergy severity score (FASS) with ordinal (oFASS) and numerical (nFASS) formats. oFASS with 3 and 5 grades were generated through expert consensus, and nFASS by mathematical modeling. Evaluation was performed in the EuroPrevall outpatient clinic cohort (8232 food reactions) by logistic regression with request of emergency care and medications used as outcomes. Discrimination, classification, and calibration were calculated. Bootstrapping internal validation was followed by external validation (logistic regression) in 5 cohorts (3622 food reactions). Correlation of nFASS with the severity classification done by expert allergy clinicians by Best-Worst Scaling of 32 food reactions was calculated. Results: oFASS and nFASS map consistently, with nFASS having greater granularity. With the outcomes emergency care, adrenaline and critical medical treatment, oFASS and nFASS had a good discrimination (receiver operating characteristic area under the curve [ROC-AUC]>0.80), classification (sensitivity 0.87–0.92, specificity 0.73–0.78), and calibration. Bootstrapping over ROC-AUC showed negligible biases (1.0 × 10−6–1.23 × 10−3). In external validation, nFASS performed best with higher ROC-AUC. nFASS was strongly correlated (R 0.89) to best-worst scoring of 334 expert clinicians. Conclusion: FASS is a validated and reliable method to measure severity of food allergic reactions. The ordinal and numerical versions that map onto each other are suitable for use by different stakeholders in different settings. © 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.

Additional file 2: Table 2. Source and protein family of proteins examined in the study. Overview table which includes the legumes from which the individual proteins were purified. In addition, the protein family is indicated and the classification if the protein is either a weakly or strongly allergenic protein is indicated.

Additional file 1: Table 1. 64 DEGs shared by the two comparisons of weakly and strongly allergenic 2S albumins and 7S globulins. 64 genes that were differently expressed when PBMCs were incubated with a pair of weakly and strongly allergenic proteins from the 2S albumin protein family compared to a protein pair of weakly and strongly allergenic proteins from the 7S globulin protein family.

Additional file 4: Table 4. Multiplex gene set 2. Table containing the sequences of the primers and probes of CCL7 and RASD2 designed with primer express software (Applied Biosystems).

Länsstyrelsen i Stockholms län följer utvecklingen omkring oönskade ämnen i avloppsslam från länets tillståndspliktiga avloppsreningsverk, som en del av den regionala miljöövervakningen. Slammet är en god indikator för de ämnena som cirkulerar i samhället och som reningsverken renar bort från det inkommande vattnet. I denna rapport har data sammanställts över ett antal metaller och organiska ämnen som verksamhetsutövarna har rapporterat in till den svenska miljörapporteringsportalen för åren 2009 till och med 2019. Att allt fler människor bosätter sig i Stockholmsregionen har betydelse för reningsverkens drift, de behöver ta hand om mer spillvatten och det blir mer slam som produceras lokalt. En förskjutning från tidigare huvudsakligen industriellt spillvatten till primärt hushållsrelaterat spillvatten kan ses. Slammets användningsområden är framförallt inriktade på jordförbättringsmedel och återföring till åkermark. I Stockholms omedelbara närhet saknas tillräckligt med åkermark som har ett underskott av fosfor. För att kunna tillåta återföring till samhällets kretslopp behöver en god kemisk kvalitet av slammet säkerställas. För kvalitetshöjande arbete och kontroll av detta har VA-branschens riksorganisation, Svenskt Vatten startat REVAQ, ett kvalitetssäkringssystem för avloppsslam. Över 95 procent av länets avloppsslam produceras i de fyra största reningsverken, som alla är Revaq-certifierade. Länets produktion uppgår till en femtedel av Sveriges produktion totalt. Halterna av de undersökta metallerna kadmium, kvicksilver, bly, krom och silver har minskat successivt i slam från de sju Revaq-certifierade avloppsreningsverken i Stockholms län under perioden 2009 till och med 2019. För metallerna koppar, zink och nickel är mellanårsvariationen relativt stor medan ingen tydlig minskning eller ökning kan ses i slammet. För de undersökta organiska ämnena, PAH, PCB och nonylfenol, kan en tydlig minskning konstateras för PCB och nonylfenol under perioden 2009 till och med 2019, medan halterna av PAH i slam varken ökar eller minskar under perioden. Mellanårsvariationen för både PAH och PCB är relativt stor, mindre för nonylfenol. Vid slamåterföring på åkermark i och omkring länet finns det risk för en högre ackumulering av kadmium, kvicksilver och silver i åkermarken än den som är önskvärd, det vill säga med en kortare dubbleringstid än 500 år (större än 0,02 % ökning per år). Samtidigt minskar dessa ämnens förekomst i slammet och i det atmosfäriska nedfallet. De av Naturvårdsverket föreslagna gränsvärdena och de av VA-branschen föreslagna riktvärden kan följas för nästan allt slam som producerats i länets sju Revaq-certifierade reningsverk. För det slammet som inte klarar de uppsatta gräns- och riktvärden behöver branschen ha tillgång till alternativa metoder för användning av slampartier. Våra regionala åkermarker, i hela Mälardalen, består mestadels av styva leror med en basisk karaktär och vanligen ett överskott av fosfor. Vid spridning av oönskade metaller och svårnedbrytbara organiska ämnen på dessa jordar kan halterna öka i jorden. Det innebär inte automatiskt en större risk för miljö och hälsa, eftersom metaller eller organiska ämnen i dessa leror ligger relativt hård bundna till jorden, med relativt låg risk för läckage till yt- eller grundvatten samt upptag i grödor. Däremot kan de relativt höga halterna i jorden utgöra en risk för upptag av betande djur som kommer i kontakt med jordpartiklarna. Att återföra slam till dessa lerjordar bör egentligen snarare syfta till att förbättra jordstrukturen än att påföra ännu mer fosfor. Doseringen bör avpassas så att det förutom hållbara halter av metaller och organiska föreningar i jord och grödor, även tas större hänsyn till risken för näringsläckage till länets vatten.

Additional file 5: Figure 1. The immunological roles of CCL2, CCL7 and RASD2 in mast cell activation and food allergy. Figure explaining the immunological roles of CCL2, CCL7 and RASD2 in mast cell activation and food allergy.

Additional file 3: Table 3. Multiplex gene set 1. Table containing the sequences of the primers and probes of CCL2 and IL-24 designed with primer express software (Applied Biosystems).

Background: Walnut allergy is common across the globe, but data on the involvement of individual walnut components are scarce. Objectives: To identify geographical differences in walnut component sensitization across Europe, explore cosensitization and cross-reactivity, and assess associations of clinical and serological determinants with severity of walnut allergy. Methods: As part of the EuroPrevall outpatient surveys in 12 European cities, standardized clinical evaluation was conducted in 531 individuals reporting symptoms to walnut, with sensitization to all known walnut components assessed in 202 subjects. Multivariable Lasso regression was applied to investigate predictors for walnut allergy severity. Results: Birch-pollen–related walnut sensitization (Jug r 5) dominated in Northern and Central Europe and lipid transfer protein sensitization (Jug r 3) in Southern Europe. Profilin sensitization (Jug r 7) was prominent throughout Europe. Sensitization to storage proteins (Jug r 1, 2, 4, and 6) was detected in up to 10% of subjects. The walnut components that showed strong correlations with pollen and other foods differed between centers. The combination of determinants best predicting walnut allergy severity were symptoms upon skin contact with walnut, atopic dermatitis (ever), family history of atopic disease, mugwort pollen allergy, sensitization to cat or dog, positive skin prick test result to walnut, and IgE to Jug r 1, 5, 7, or carbohydrate determinants (area under the curve = 0.81; 95% CI, 0.73-0.89). Conclusions: Walnut-allergic subjects across Europe show clear geographical differences in walnut component sensitization and cosensitization patterns. A predictive model combining results from component-based serology testing with results from extract-based testing and information on clinical background allows for good discrimination between mild to moderate and severe walnut allergy. © 2020 The Authors

Contains fulltext : 237755.pdf (Publisher’s version ) (Open Access) BACKGROUND: Evidence on safety and effectiveness of omalizumab for treatment of chronic urticaria in pediatric patients is scarce and limited to case reports. In particular, drug survival of omalizumab has not yet been investigated, which is a key element in the evaluation of its clinical performance. The aim of this study was to investigate safety, effectiveness, and drug survival rates of omalizumab in a daily practice cohort of pediatric patients with chronic urticaria (CU). METHODS: This is a multicenter study including all pediatric patients from an academic center (Wilhelmina Children's Hospital) and a general center (Diakonessenhuis Hospital) in the Netherlands, who started omalizumab treatment before the age of 18 years. Data on safety, effectiveness, time to discontinuation, and reasons for discontinuation of treatment were assessed. Drug survival of omalizumab was estimated using the Kaplan-Meier survival analysis. RESULTS: A total of 38 patients, who started treatment between January 2014 and January 2020, were included. Most patients (68.4%) used omalizumab without reporting any side effects and a complete or good response to treatment was achieved in 76.3% of patients. The 1- and 2-year drug survival rates were 62% and 50%, respectively, with well-controlled disease activity as the most frequent reason for discontinuation in 69.2% of patients, followed by ineffectiveness in 23.1% and side effects in 7.7% of patients. CONCLUSIONS: This study demonstrates high safety and effectiveness of omalizumab treatment in pediatric patients with CU, which will aid clinical decision making and management of expectations when choosing omalizumab treatment for pediatric patients with CU.