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Background: Studies of human IgE and its targeted epitopes on allergens have been very limited. We have an established method to immortalize IgE encoding B cells from allergic individuals., Objective: To develop an unbiased and comprehensive panel of peanut-specific human IgE mAbs to characterize key immunodominant antigenic regions and epitopes on peanut allergens to map the molecular interactions responsible for inducing anaphylaxis., Methods: Using human hybridoma technology to immortalize IgE encoding B cells from peripheral blood of subjects with severe peanut allergy, we generated a panel of naturally occurring human IgE mAbs in an unbiased manner. Isolated IgE mAbs were characterized extensively in allergen binding assays, peptide array analysis, antigenic mapping, binding kinetic analysis, serum blocking, skin testing inhibition, and functional assessment using human FcεRI transgenic mice., Results: We created a large panel of 54 peanut-specific IgE mAbs, of which 63% were specific for Ara h 2 and/or 6. Pairs of IgE mAbs with the same antigen-specificity but different binding sites were able to mediate passive systemic anaphylaxis in FcεRI transgenic mice. A single mAb targeting the repetitive motif on Ara h 2 was able to induce degranulation and anaphylaxis on its own. IgG1 switched-variant immunoglobulins of the IgE mAb inhibited patients´ IgE binding to peanut extract between 30-60% (ImmunoCAP) and reduced peanut extract-induced skin wheal sizes by 1.6-7.4 millimeters in peanut allergic patients., Conclusion: We created a molecular map of the IgE antibody response to the most important peanut allergen proteins to enable the design of new allergy immunotherapies and vaccines., (Copyright © 2025. Published by Elsevier Inc.)

Background: Solubility is a common feature of allergens. However, the causative relationship between this protein-intrinsic feature and sensitization capacity of allergens is not fully understood. This study aimed to proof the concept of solubility as a protein intrinsic feature of allergens., Methods: The soluble birch pollen allergen Bet v 1 was covalently coupled to 1 μm silica particles. IgE-binding and -cross-linking capacity was assessed by inhibition ELISA and mediator release assay, respectively. Alterations in adjuvanticity by particle-loading were investigated by activation of dendritic cells, mast cells and the Toll-like receptor 4 pathway as well as by Th2 polarization in an IL-4 reporter mouse model. In BALB/c mice, particle-loaded and soluble Bet v 1 were compared in a model of allergic sensitization. Antigen uptake and presentation was analysed by restimulating human Bet v 1-specific T cell lines., Results: Covalent coupling of Bet v 1 to silica particles resulted in an insoluble antigen with retained IgE-binding and -cross-linking capacity and no increase in adjuvanticity. In vivo, particle-loaded Bet v 1 induced significantly lower Bet v 1-specific (s)IgE, whereas sIgG1 and sIgG2a levels remained unaffected. The ratio of Th2 to Th1 cells was significantly lower in mice sensitized with particle-loaded Bet v 1. Particle-loading of Bet v 1 resulted in a 24-fold higher T cell activation capacity in Bet v 1-specific T cell lines, indicating more efficient uptake and presentation than of soluble Bet v 1., Conclusions: Our results show that solubility is a decisive factor contributing to the sensitization capacity of allergens. The reduction in sensitization capacity of insoluble, particle-loaded antigens results from enhanced antigen uptake and presentation compared to soluble allergens., (Copyright © 2024 Japanese Society of Allergology. Published by Elsevier B.V. All rights reserved.)

Background: Allergen-carrying virus-like particles are effective and safe means of allergen immunotherapy (AIT) in rodent models., Objective: To study the development of allergen-blocking immunoglobulin (Ig)G in dogs injected with Der f 2-carrying enveloped plant-based bioparticles (eBPs)., Materials and Methods: Laboratory beagle dogs were injected intradermally (ID) or subcutaneously (SC) with Der f 2-eBP three times at 2-week intervals. A basophil mediator release assay was used to compare the reactivity of Der f 2-eBPs to that of recombinant Der f 2. Allergen-specific IgG serum levels were determined by immunoblotting and ELISA. The allergen-blocking potential of postvaccination IgG was assessed by Pet Allergy Xplorer (PAX) macroarray and basophil mediator release inhibition assays., Results: The amount of Der f 2 eBPs needed to induce basophil activation was 1000-fold higher than that of the soluble natural allergen. In both immunisation groups, eBP injections caused no adverse events and induced Der f 2-specific IgG, first detected on Day (D)14 and peaking on D41. The co-incubation of sera with a Der f 2-IgE-rich canine serum pool resulted in a mean PAX inhibition of 70% (ID) to 80% (SC) on D41. For both groups, the inhibition of basophil mediator release reached 75% on D28 and D41. The percentage inhibition of PAX and mediator release correlated significantly with Der f 2 IgG levels., Conclusion and Clinical Relevance: Intradermal and subcutaneous injections of Der f 2-eBPs were safe and increased Der f 2-specific IgG. The clinical benefit of immunotherapy will be evaluated in future trials enrolling atopic dogs allergic to house dust mites., (© 2024 ESVD and ACVD.)

Pollen traps, the current gold standard to determine pollen load and thereby the allergy season, are not sufficient to determine the allergenic risk. Therefore, the establishment of highly sensitive assays for allergen measurement is of highest interest. Herein, a graphene field-effect transistor (GFET) was constructed on an interdigitated electrodes chip to develop an immersible biosensor, which was used to detect the major birch pollen allergen Bet v 1. Graphene was wet-transferred on interdigitated electrodes that contain a reference electrode used as a liquid gate in the GFET. Using a standard ELISA protocol, two different anti-Bet v 1 antibodies were chosen and immobilized on graphene for the specific capture of the target allergen. The sensitivity of the GFET biosensor was evaluated using a standard Ag/AgCl liquid gate electrode and a reference electrode when the chip was immersed in Bet v 1-containing solutions. The results showed a higher performance and sensitivity for Bet v 1 detection compared to a mediator release method, one of the most sensitive assays for allergen detection. Compared with conventional methods of allergen detection, these immersible biosensors significantly improved the speed and level of detection providing the foundation of a point-of-need platform for in-field application. Furthermore, the proposed technique provides both a new biosensor for allergen detection and a strategy for designing low-cost integrated biosensors., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

This study examined the acute effects of exercise testing on immunology markers, established blood-based biomarkers, and questionnaires in endurance athletes, with a focus on biological sex differences. Twenty-four healthy endurance-trained participants (16 men, age: 29.2± 7.6 years, maximal oxygen uptake ( V ˙ O 2 max ): 59.4 ± 7.5 ml · min -1 · kg -1 ; 8 women, age: 26.8 ± 6.1 years, V ˙ O 2 max : 52.9 ± 3.1 ml · min -1 · kg -1 ) completed an incremental submaximal exercise test and a ramp test. The study employed exploratory bioinformatics analysis: mixed ANOVA, k-means clustering, and uniform manifold approximation and projection, to assess the effects of exhaustive exercise on biomarkers and questionnaires. Significant increases in biomarkers (lymphocytes, platelets, procalcitonin, hemoglobin, hematocrit, red blood cells, cell-free DNA (cfDNA)) and fatigue were observed post-exercise. Furthermore, differences pre- to post-exercise were observed in cytokines, cfDNA, and other blood biomarkers between male and female participants. Three distinct groups of athletes with differing proportions of females (Cluster 1: 100% female, Cluster 2: 85% male, Cluster 3: 37.5% female and 65.5% male) were identified with k-means clustering. Specific biomarkers (e.g., interleukin-2 (IL-2), IL-10, and IL-13, as well as cfDNA) served as primary markers for each cluster, potentially informing individualized exercise responses. In conclusion, our study identified exercise-sensitive biomarkers and provides valuable insights into the relationships between biological sex and biomarker responses., Competing Interests: The study has received funding from the Red Bull Athlete Performance Center. The funding body has not peer-reviewed the manuscript. This sponsor was not involved in the study design, the writing of the manuscript, or the decision to submit the manuscript for publication., (Copyright © Institute of Sport – National Research Instutite.)

Immunity to Ascaris lumbricoides influences the pathogenesis of allergic diseases. Antibody responses to its proteins have been found to be associated with asthma presentation; however, helminth products that induce immunosuppression have been reported, which also raise specific antibodies. We aimed to evaluate antibody responses (IgE, IgG4 and IgG) to two A. lumbricoides molecules, Asc l 5 and Al-CPI (an anti-inflammatory Cysteine Protease Inhibitor), in an endemic population, exploring their relationships with the infection and asthma. The two molecules were produced as recombinant proteins in E. coli expression systems. Specific antibodies were detected by ELISA. Lower human IgE, but higher IgG4 and IgG antibody levels were observed for Al-CPI than for rAsc l 5. The IgE/IgG4 isotype ratio was significantly higher for Asc l 5 than for Al-CPI. In humans Al-CPI did not induce basophil activation as has been previously described for Asc l 5. In mice, Al-CPI induced fewer IgE responses, but more IgG2a antibody titers than rAsc l 5. Our results suggest that these molecules elicit different patterns of immune response to A. lumbricoides .

Introduction: Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity., Materials and Methods: The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3-18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing β -hexosaminidase release from a humanized rat basophilic cell line., Results: Human IgE monoclonal antibodies ( n  = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450-1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of β -hexosaminidase (35%-80%) from a humanized rat basophilic cell line., Conclusions: Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes., Competing Interests: The research approach was developed by several authors who are employees of InBio (BS, KR, MB, SA, JG, AP, and MC). The hIgE mAb and some allergens used in this study were produced by InBio. AP is a contact principal investigator of the NIH R01 award that provided funding for the study. MC has a financial interest in InBio and is a co-investigator for the NIH R01 award. InBio has a license agreement with Vanderbilt University Medical Center for the commercialization of the hIgE mAb for research and diagnostic purposes. The hIgE mAb covered by this agreement are available from InBio (www.inbio.com). SS is an inventor on US patent 10908168-B2 for the generation of human IgE monoclonal antibodies, has received patent royalties, and has related patents pending. RR is a consultant for HAL Allergy BV, Citeq BV, Angany Inc., Reacta Healthcare Ltd., Mission MightyMe, and AB Enzymes; receives speaker fees from HAL Allergy BV, ALK, and Thermo Fisher Scientific; and has stock options with Angany Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2023 Smith, Reid Black, Bermingham, Agah, Glesner, Versteeg, van Ree, Pena-Amelunxen, Aglas, Smith, Pomés and Chapman.)

Toll-like receptors of the human immune system are specialized pathogen detectors able to link innate and adaptive immune responses. TLR ligands include among others bacteria-, mycoplasma- or virus-derived compounds such as lipids, lipo- and glycoproteins and nucleic acids. Not only are genetic variations in TLR-related genes associated with the pathogenesis of allergic diseases, including asthma and allergic rhinitis, their expression also differs between allergic and non-allergic individuals. Due to a complex interplay of genes, environmental factors, and allergen sources the interpretation of TLRs involved in immunoglobulin E-mediated diseases remains challenging. Therefore, it is imperative to dissect the role of TLRs in allergies. In this review, we discuss i) the expression of TLRs in organs and cell types involved in the allergic immune response, ii) their involvement in modulating allergy-associated or -protective immune responses, and iii) how differential activation of TLRs by environmental factors, such as microbial, viral or air pollutant exposure, results in allergy development. However, we focus on iv) allergen sources interacting with TLRs, and v) how targeting TLRs could be employed in novel therapeutic strategies. Understanding the contributions of TLRs to allergy development allow the identification of knowledge gaps, provide guidance for ongoing research efforts, and built the foundation for future exploitation of TLRs in vaccine design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wenger, Grosse-Kathoefer, Kraiem, Pelamatti, Nunes, Pointner and Aglas.)

Background: Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated., Methods: Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release., Results: One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization., Conclusion: The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb., Competing Interests: The authors declare that this study received funding from InBio, Charlottesville, VA, USA. The funder had the following involvement in the study: InBio scientists (BS, AP and MC) contributed to the study design and writing the paper. The hIgE mAb and some of the allergens used in this study were provided by InBio. AP is an employee of InBio and the contact principal investigator of the NIH R01 award that provided funding for the study. MC has a financial interest in InBio and is a coinvestigator on the NIH R01 award. InBio has a license agreement with Vanderbilt University Medical Center for commercialization of hIgE mAb for research and diagnostic purposes. The hIgE mAb covered by this agreement are available from InBio www.inbio.com. SS is an inventor on U.S. patent 10908168-B2 for generation of human IgE monoclonal antibodies, has received patent royalties and has related patents pending. RR has consultancies in: HAL Allergy, Citeq, Angany Inc., Mission MightyMe, AB Enzymes, Reacta Healthcare, The Protein Brewery; speaker fees for HAL Allergy, Thermo Fisher Scientific,ALK; and stock options: Angany Inc., (Copyright © 2023 Pena-Castellanos, Smith, Pomés, Smith, Stigler, Widauer, Versteeg, van Ree, Chapman and Aglas.)

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure., (© 2023 The Authors. Pediatric Allergy and Immunology published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: In birch-dominated areas, allergies to pollen from trees of the order Fagales are considered to be initiated by the major birch pollen allergen Bet v 1. However, the sensitizing activity of Bet v 1-homologs in Fagales pollen might be underestimated. Allergen-specific T-cells are crucial in the sensitization process. The T-cell response to major allergens from alder, hazel, oak, hornbeam, chestnut, beech, and chestnut pollen has not yet been analyzed. Here, we characterized the cellular cross-reactivity of major allergens in Fagales pollen with Bet v 1., Methods: T-cell-lines (TCL) were established from allergic individuals with Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1, and Que a 1, and tested for reactivity with Bet v 1 and synthetic overlapping 12-mer peptides representing its primary sequence. Aln g 1-specific TCL was additionally tested with Aln g 1-derived peptides and all allergens. IgE-competition experiments with Aln g 1 and Bet v 1 were performed., Results: T-cell-lines initiated with Fagales pollen allergens varied strongly in their reactivity with Bet v 1 and by the majority responded stronger to the original stimulus. Cross-reactivity was mostly restricted to the epitope Bet v 1 142-153 . No distinct cross-reactivity of Aln g 1-specific T-cells with Bet v 1 was detected. Among 22 T-cell epitopes, Aln g 1 contained two immunodominant epitopes. Bet v 1 inhibited IgE-binding to Aln g 1 less potently than Aln g 1 itself., Conclusion: The cellular cross-reactivity of major Fagales pollen allergens with Bet v 1 was unincisive, particularly for Aln g 1, most akin to Bet v 1. Our results indicate that humoral and cellular responses to these allergens are not predominantly based on cross-reactivity with the major birch pollen allergen but suggest a Bet v 1-independent sensitization in individuals from birch tree-dominated areas., (© 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

The incorporation of nanomaterials into consumer products has substantially increased in recent years, raising concerns about their safety. The inherent physicochemical properties of nanoparticles allow them to cross epithelial barriers and gain access to immunocompetent cells. Nanoparticles in cosmetic products can potentially interact with environmental allergens, forming a protein corona, and together penetrate through damaged skin. Allergen-nanoparticle interactions may influence the immune response, eventually resulting in an adverse or beneficial outcome in terms of allergic reactivity. This study determines the impact of silica nanoparticle-allergen interactions on allergic sensitization by studying the major molecular mechanisms affecting allergic responses. The major birch pollen allergen Bet v 1 was chosen as a model allergen and the birch pollen extract as a comparator. Key events in immunotoxicity including allergen uptake, processing, presentation, expression of costimulatory molecules and cytokine release were studied in human monocyte-derived dendritic cells. Using an in vivo sensitization model, murine Bet v 1-specific IgG and IgE levels were monitored. Upon the interaction of allergens with silica nanoparticles, we observed an enhanced uptake of the allergen by macropinocytosis, improved proteolytic processing, and presentation concomitant with a propensity to increase allergen-specific IgG2a and decrease IgE antibody levels. Together, these events suggest that upon nanoparticle interactions the immune response is biased towards a type 1 inflammatory profile, characterized by the upregulation of T helper 1 (Th1) cells. In conclusion, the interaction of the birch pollen allergen with silica nanoparticles will not worsen allergic sensitization, a state of type 2-inflammation, but rather seems to decrease it by skewing towards a Th1-dominated immune response.

Although aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous allergen immunotherapy (SCIT). Our objective was to evaluate allergen-bearing liposomes as possible alternative for alum-adsorption in SCIT. A self-assembling, coiled-coil forming peptide pair was used to anchor the major birch pollen allergen Bet v 1 to the surface of cationic liposomes. The resulting nanoparticulate liposomes were characterized with respect to their physicochemical, allergenic and immunological properties. Allergenicity was studied by ImmunoCAP inhibition and rat basophil leukemia (RBL) cell assays. Immunogenicity (immunoglobulin responses) and immune skewing (cytokine responses) were evaluated upon immunization of naïve mice, and compared to alum-adsorbed Bet v 1. Bet v 1-bearing cationic liposomes with a diameter of ∼200 nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG 1 /IgG 2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT., Competing Interests: HW and HS are employees of HAL Allergy BV. RR is a consultant for HAL Allergy BV, Citeq BV, Reacta Healthcare Ltd., Angany Inc., Mission MightyMe and AB Enzymes GmbH. RR reports speaker fees from HAL Allergy BV, ThermoFisher Scientific and ALK and has stock options of Angany Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Warmenhoven, Leboux, Bethanis, van Strien, Logiantara, van Schijndel, Aglas, van Rijt, Slütter, Kros, Jiskoot and van Ree.)

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT., Competing Interests: HW is and JWS was an employee of HAL Allergy BV. JWS is an employee of SeraNovo BV. RR is a consultant for HAL Allergy BV, Citeq BV, Reacta Healthcare Ltd., Angany Inc., Mission MightyMe and AB Enzymes GmbH. RR reports speaker fees from HAL Allergy BV, ThermoFisher Scientific and ALK and has stock options of Angany Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van Strien, Warmenhoven, Logiantara, Makurat, Aglas, Bethanis, Leboux, van Rijt, MacKay, van Schijndel, Schneider, Olsthoorn, Jiskoot, van Ree and Kros.)

Load management, i.e., prescribing, monitoring, and adjusting training load, is primarily aimed at preventing injury and maximizing performance. The search for objective monitoring tools to assess the external and internal load of athletes is of great interest for sports science research. In this 4-week pilot study, we assessed the feasibility and acceptance of an extensive monitoring approach using biomarkers, neuromuscular performance, and questionnaires in an elite youth soccer setting. Eight male players (mean ± SD: age: 17.0 ± 0.6 years, weight: 69.6 ± 8.2 kg, height: 177 ± 7 cm, VO 2max : 62.2 ± 3.8 ml/min/kg) were monitored with a local positioning system (e.g., distance covered, sprints), biomarkers (cell-free DNA, creatine kinase), questionnaires, neuromuscular performance testing (counter-movement jump) and further strength testing (Nordic hamstring exercise, hip abduction and adduction). Feasibility was high with no substantial impact on the training routine and no adverse events such as injuries during monitoring. Adherence to the performance tests was high, but adherence to the daily questionnaires was low, and decreased across the study period. Occasional significant correlations were observed between questionnaire scores and training load data, as well as between questionnaire scores and neuromuscular performance. However, due to the small sample size, these findings should be treated with caution. These preliminary results highlight the feasibility of the approach in elite soccer, but also indicate that modifications are needed in further large-scale studies, particularly in relation to the length of the questionnaire., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Haller, Blumkaitis, Strepp, Schmuttermair, Aglas, Simon, Neuberger, Kranzinger, Kranzinger, O’Brien, Ergoth, Raffetseder, Fail, Düring and Stöggl.)

Background: Performing multiple high-intensity interval training (HIIT) sessions in a compressed period of time (approximately 7-14 days) is called a HIIT shock microcycle (SM) and promises a rapid increase in endurance performance. However, the efficacy of HIIT-SM, as well as knowledge about optimal training volumes during a SM in the endurance-trained population have not been adequately investigated. This study aims to examine the effects of two different types of HIIT-SM (with or without additional low-intensity training (LIT)) compared to a control group (CG) on key endurance performance variables. Moreover, participants are closely monitored for stress, fatigue, recovery, and sleep before, during and after the intervention using innovative biomarkers, questionnaires, and wearable devices., Methods: This is a study protocol of a randomized controlled trial that includes the results of a pilot participant. Thirty-six endurance trained athletes will be recruited and randomly assigned to either a HIIT-SM (HSM) group, HIIT-SM with additional LIT (HSM + LIT) group or a CG. All participants will be monitored before (9 days), during (7 days), and after (14 days) a 7-day intervention, for a total of 30 days. Participants in both intervention groups will complete 10 HIIT sessions over 7 consecutive days, with an additional 30 min of LIT in the HSM + LIT group. HIIT sessions consist of aerobic HIIT, i.e., 5 × 4 min at 90-95% of maximal heart rate interspersed by recovery periods of 2.5 min. To determine the effects of the intervention, physiological exercise testing, and a 5 km time trial will be conducted before and after the intervention., Results: The feasibility study indicates good adherence and performance improvement of the pilot participant. Load monitoring tools, i.e., biomarkers and questionnaires showed increased values during the intervention period, indicating sensitive variables., Conclusion: This study will be the first to examine the effects of different total training volumes of HIIT-SM, especially the combination of LIT and HIIT in the HSM + LIT group. In addition, different assessments to monitor the athletes' load during such an exhaustive training period will allow the identification of load monitoring tools such as innovative biomarkers, questionnaires, and wearable technology., Trial Registration: clinicaltrials.gov, NCT05067426. Registered 05 October 2021-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05067426 . Protocol Version Issue date: 1 Dec 2021. Original protocol. Authors: TLS, NH., (© 2022. The Author(s).)

B cells are key players in the mechanisms underlying allergic sensitization, allergic reactions, and tolerance to allergens. Allergen-specific immune responses are initiated when peptide:MHCII complexes on dendritic cells are recognized by antigen-specific receptors on T cells followed by interactions between costimulatory molecules on the surfaces of B and T cells. In the presence of IL-4, such T-B cell interactions result in clonal expansion and isotype class-switching to IgE in B cells, which will further differentiate into either memory B cells or PCs. Allergic reactions are then triggered upon cross-linking of IgE-FcɛRI complexes on basophils and mast cells, leading to cell degranulation and the release of pro-inflammatory mediators.Mechanisms underlying effective allergen-specific immunotherapy (AIT) involve the induction of Tregs and the secretion of blocking IgG4 antibodies, which together mediate the onset and maintenance of immune tolerance towards non-hazardous environmental antigens. However, the importance of regulatory B cells (Breg) for tolerance induction during AIT has gained more attention lately. Studies in grass pollen- and house dust mite-allergic patients undergoing SCIT reported increased frequencies of IL-10+ Breg cells and a positive correlation between their number and the improvement of clinical symptoms. Thus, Breg are emerging as biomarkers for monitoring tolerance to allergens under natural exposure conditions and during AIT. Further research on the role of other anti-inflammatory cytokines secreted by Breg will help to understand their role in disease development and tolerance induction., (© 2021. Springer Nature Switzerland AG.)

Background: Evidence has accumulated that birch pollen immunotherapy reduces rhinoconjunctivitis to pollen of birch homologous trees. Therapeutic efficacy has been associated with IgE-blocking IgG antibodies. We have recently shown that sera collected after 16 weeks of sublingual immunotherapy with recombinant Bet v 1 (rBet v 1-SLIT) display strong IgE-blocking bioactivity for Bet v 1. Here, we assessed whether rBet v 1-SLIT-induced IgG antibodies display cross-blocking activity to related allergens in Fagales pollen., Methods: IgE, IgG1 and IgG4 reactivity to recombinant Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1 and Que a 1 were assessed in pre- and post-SLIT samples of 17 individuals by ELISA. A basophil inhibition assay using stripped basophils re-sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to allergens after incubation with pre-SLIT or post-SLIT samples. IgG1 and IgG4 were depleted from post-SLIT samples to assess its contribution to IgE-cross-blocking., Results: Sublingual immunotherapy with recombinant Bet v 1 boosted cross-reactive IgE antibodies and induced IgG1 and IgG4 antibodies with inter- and intra-individually differing reactivity to the homologs. Highly variable cross-blocking activities of post-SLIT samples to the different allergens were found. IgG1 and IgG4 antibodies displayed cross-blocking activity with individual variance., Conclusions: Our mechanistic approach suggested that immunotherapy with the reference allergen Bet v 1 induces individual repertoires of cross-reactive IgG1 and IgG4 antibodies. The cross-blocking bioactivity of these antibodies was also highly variable and neither predictable from protein homology nor IgE-cross-reactivity., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (FcεRI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of β-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Background: Common ragweed has been spreading as a neophyte in Europe. Elevated CO 2 levels, a hallmark of global climate change, have been shown to increase ragweed pollen production, but their effects on pollen allergenicity remain to be elucidated., Methods: Ragweed was grown in climate-controlled chambers under normal (380 ppm, control) or elevated (700 ppm, based on RCP4.5 scenario) CO 2 levels. Aqueous pollen extracts (RWE) from control- or CO 2 -pollen were administered in vivo in a mouse model for allergic disease (daily for 3-11 days, n = 5) and employed in human in vitro systems of nasal epithelial cells (HNECs), monocyte-derived dendritic cells (DCs), and HNEC-DC co-cultures. Additionally, adjuvant factors and metabolites in control- and CO 2 -RWE were investigated using ELISA and untargeted metabolomics., Results: In vivo, CO 2 -RWE induced stronger allergic lung inflammation compared to control-RWE, as indicated by lung inflammatory cell infiltrate and mediators, mucus hypersecretion, and serum total IgE. In vitro, HNECs stimulated with RWE increased indistinctively the production of pro-inflammatory cytokines (IL-8, IL-1β, and IL-6). In contrast, supernatants from CO 2 -RWE-stimulated HNECs, compared to control-RWE-stimulated HNECS, significantly increased TNF and decreased IL-10 production in DCs. Comparable results were obtained by stimulating DCs directly with RWEs. The metabolome analysis revealed differential expression of secondary plant metabolites in control- vs CO 2 -RWE. Mixes of these metabolites elicited similar responses in DCs as compared to respective RWEs., Conclusion: Our results indicate that elevated ambient CO 2 levels elicit a stronger RWE-induced allergic response in vivo and in vitro and that RWE increased allergenicity depends on the interplay of multiple metabolites., (© 2020 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides .

Phospholipase A 2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA 2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA 2 inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA 2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA 2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA 2 activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (10 4 to 10 6 ) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT., (Copyright © 2020 Aglas, Bethanis, Chrusciel, Stolz, Gruen, Jaakkola, Jongejan, Yatkin and Van Ree.)

Background: Pollen exposure induces local and systemic allergic immune responses in sensitized individuals, but nonsensitized individuals also are exposed to pollen. The kinetics of symptom expression under natural pollen exposure have never been systematically studied, especially in subjects without allergy., Objective: We monitored the humoral immune response under natural pollen exposure to potentially uncover nasal biomarkers for in-season symptom severity and identify protective factors., Methods: We compared humoral immune response kinetics in a panel study of subjects with seasonal allergic rhinitis (SAR) and subjects without allergy and tested for cross-sectional and interseasonal differences in levels of serum and nasal, total, and Betula verrucosa 1-specific immunoglobulin isotypes; immunoglobulin free light chains; cytokines; and chemokines. Nonsupervised principal component analysis was performed for all nasal immune variables, and single immune variables were correlated with in-season symptom severity by Spearman test., Results: Symptoms followed airborne pollen concentrations in subjects with SAR, with a time lag between 0 and 13 days depending on the pollen type. Of the 7 subjects with nonallergy, 4 also exhibited in-season symptoms whereas 3 did not. Cumulative symptoms in those without allergy were lower than in those with SAR but followed the pollen exposure with similar kinetics. Nasal eotaxin-2, CCL22/MDC, and monocyte chemoattactant protein-1 (MCP-1) levels were higher in subjects with SAR, whereas IL-8 levels were higher in subjects without allergy. Principal component analysis and Spearman correlations identified nasal levels of IL-8, IL-33, and Betula verrucosa 1-specific IgG 4 (sIgG 4 ) and Betula verrucosa 1-specific IgE (sIgE) antibodies as predictive for seasonal symptom severity., Conclusions: Nasal pollen-specific IgA and IgG isotypes are potentially protective within the humoral compartment. Nasal levels of IL-8, IL-33, sIgG 4 and sIgE could be predictive biomarkers for pollen-specific symptom expression, irrespective of atopy., (Copyright © 2020. Published by Elsevier Inc.)

The mechanisms involved in the induction of allergic sensitization by pollen are not fully understood. Within the last few decades, findings from epidemiological and experimental studies support the notion that allergic sensitization is not only dependent on the genetics of the host and environmental factors, but also on intrinsic features of the allergenic source itself. In this review, we summarize the current concepts and newest advances in research focusing on the initial mechanisms inducing pollen sensitization. Pollen allergens are embedded in a complex and heterogeneous matrix composed of a myriad of bioactive molecules that are co-delivered during the allergic sensitization. Surprisingly, several purified allergens were shown to lack inherent sensitizing potential. Thus, growing evidence supports an essential role of pollen-derived components co-delivered with the allergens in the initiation of allergic sensitization. The pollen matrix, which is composed by intrinsic molecules ( e.g. proteins, metabolites, lipids, carbohydrates) and extrinsic compounds ( e.g. viruses, particles from air pollutants, pollen-linked microbiome), provide a specific context for the allergen and has been proposed as a determinant of Th2 polarization. In addition, the involvement of various pattern recognition receptors (PRRs), secreted alarmins, innate immune cells, and the dependency of DCs in driving pollen-induced Th2 inflammatory processes suggest that allergic sensitization to pollen most likely results from particular combinations of pollen-specific signals rather than from a common determinant of allergenicity. The exact identification and characterization of such pollen-derived Th2-polarizing molecules should provide mechanistic insights into Th2 polarization and pave the way for novel preventive and therapeutic strategies against pollen allergies., Competing Interests: Competing interestsFF reports personal fees from HAL Allergy, from the Swiss Institute of Allergy and Asthma Research (SIAF), from AllergenOnline, all outside the submitted work. All other authors declare that they have no competing interests., (© The Author(s) 2020.)

Introduction: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens., Aim: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis., Methods: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments., Results: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens., Conclusions and Clinical Relevance: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed., (© 2020 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Purpose of Review: Pathogenesis-related class 10 (PR-10) proteins are highly conserved plant proteins, which are induced in response to abiotic and biotic stress factors. To date, no unique biological function could be assigned to them. Rather a more general role of PR-10 in plant development and defense mechanisms has been proposed. In addition, some PR-10 proteins act as allergens by triggering allergic symptoms in sensitized individuals. Regardless of the diversity of reported activities, all PR-10 proteins share a common fold characterized by a solvent-accessible hydrophobic cavity, which serves as a binding site for a myriad of small-molecule ligands, mostly phytohormones and flavonoids., Recent Findings: Most of available data relate to the ligand binding activity of allergenic PR-10, particularly for those belonging to Bet v 1 family of allergens. Bet v 1 and its homologues were shown to bind flavonoids with high affinity, but the specificity appears to differ between homologues from different species. The flavonoid Q3O-(Glc)-Gal was shown to specifically bind to hazelnut Cor a 1 but not to Bet v 1. Similarly, Q3OS bound only to the major isoform Bet v 1.0101 and not to other closely related isoforms. In contrast, Bet v 1 and hazelnut Cor a 1 showed very similar binding behavior towards other flavonoids such as quercetin, genistein, apigenin, daidzein, and resveratrol. Recent research findings highlighted the importance of more precise knowledge of ligand binding for understanding the functional diversification of PR-10 proteins.

Background: Transforming growth factor β1 (TGFβ1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFβ1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFβ1 mimetic peptide (TGFβ1-mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens., Methods: The in vitro action of TGFβ1-mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL-4 reporter mice., Results: In vitro, TGFβ1-mim downregulated TNF-α production, IL-8 gene expression, and cytokine secretion, upregulated IL-10 secretion, and inhibited Phl p 5-induced basophil degranulation. During Phl p 5 sensitization in mice, TGFβ1-mim downregulated IL-2, IL-4, IL-5, IL-13, and IFN-γ, upregulated IL-10, and induced Treg cell production. Furthermore, mice treated with TGFβ1-mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization., Conclusion: The TGFβ1-mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen-specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens., (© 2019 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Background: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity., Methods: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs., Results: We identified E 1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E 1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity., Conclusion: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes., (© 2019 The Authors. Allergy published by John Wiley & Sons Ltd.)

Purpose: We set out to investigate the effectiveness of grounded sleeping on the time course of recovery with respect to muscle soreness and athletic performance after intensive eccentric muscle loading. Methods: Twenty-two healthy participants were recruited for this study and randomly assigned to an experimental group (GRD, grounded sleeping, n = 12) or control group (UGD, sham-grounded sleeping, n = 10) to evaluate the effects of 10 days recovery with GRD vs. UGD following a single intensive downhill treadmill intervention in a triple-blinded (participant, tester, and data analyst) manner. To operationalize recovery a test battery was performed at baseline and on days 1, 2, 3, 5, 7, and 10 post-intervention: (1) perception of muscle soreness (VAS), (2) creatine kinase blood levels (CK), (3) maximum voluntary isometric contraction (MVIC) for both legs, (4) counter movement jump (CMJ) and drop jump (DJ) performance. Furthermore, in four participants blood was sampled for detailed analysis of complete blood counts and serum-derived inflammation markers. Results: The downhill treadmill running intervention led to distinct changes in all measured parameters related to fatigue. These changes were detectable already 5-min post intervention and were not fully recovered 10 days post intervention. GRD led to less pronounced decrease in performance (CMJ, MVIC) and less increase with respect to CK compared with UGD (all P < 0.05). Detailed blood samples demonstrated that grounded sleeping modulates the recovery process by (a) keeping a constant hemoconcentration, as represented by the number of erythrocytes, and the hemoglobin/hematocrit values; and (b) by the reduction of muscle damage-associated inflammation markers such as, IP-10, MIP-1α, and sP-Selectin. Conclusion: The downhill running protocol is a feasible methodology to produce long term muscle soreness and muscular fatigue. GRD was shown to result in faster recovery and/or less pronounced markers of muscle damage and inflammation. GRD might be seen as a simple methodology to enhance acute and long-term recovery after intensive eccentric exercises.

Background: The clinical benefit of allergen-specific immunotherapy (AIT) involves induction of blocking antibodies. It is not clear if these antibodies function via steric hindrance alone or a combination of levels, avidities, and epitope specificities, and clinical outcome cannot be predicted. We aim to in-depth characterize serum antibody profiles during birch pollen AIT, investigate therapy-induced antibodies for their capacity to block IgE binding to Bet v 1 and correlate data with clinical outcomes., Methods: Immune responses of five birch pollen allergic patients were monitored during the first year of AIT by nasal provocation tests (NPTs), ImmunoCAP, immunoblots, direct and avidity enzyme-linked immunosorbent assays, mediator release assays, facilitated antigen binding (FAB) assays, and inhibition mediator release assays., Results: There was no correlation between NPT results and therapy-induced changes in levels (IgE, IgG, IgA, IgM), avidities, or mediator release potency of Bet v 1-specific antibodies. In FAB assays, blocking antibodies initiated upon AIT were shown to prevent formation of Bet v 1-IgE complexes of an indicator serum pool and significantly correlated with clinical readout. Inhibition mediator release assays using patient-specific IgE for passive sensitization revealed therapy-induced blocking capacities with very good correlation to NPT results. Notably, this assay was the only one to detect a non-responder during treatment in this pilot study., Conclusions: Clinical outcome of AIT depends on induction of blocking antibodies able to prevent the patient's own IgE from allergen binding. Monitoring of clinical efficacy seems to be best achieved using the inhibition mediator release assay, as development of relevant blocking antibodies can be verified in a patient-tailored manner.